Unitary Biochip Providing Sample-in to Results-out Processing and Methods of Manufacture

ABSTRACT

A biochip for the integration of all steps in a complex process from the insertion of a sample to the generation of a result, performed without operator intervention includes microfluidic and macrofluidic features that are acted on by instrument subsystems in a series of scripted processing steps. Methods for fabricating these complex biochips of high feature density by injection molding are also provided.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.13/228,263 filed on Sep. 8, 2011, which is a continuation in part ofU.S. patent application Ser. No. 13/044,485 entitled “Unitary BiochipProviding Sample-in to Results-out Processing and Methods ofManufacture” filed on Mar. 9, 2011. U.S. patent application Ser. No.13/044,485 claims priority from U.S. Provisional Application Ser. No.61/339,743 filed Mar. 9, 2010. The disclosures of U.S. patentapplication Ser. No. 13/228,263, U.S. patent application Ser. No.13/044,485 and U.S. Provisional Application Ser. No. 61/339,743 arehereby incorporated by reference in their entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under a Contract awardedby MIT Lincoln Laboratory pursuant to RFP 800002471. The government mayhave certain rights in this invention.

BACKGROUND OF THE INVENTION

The field of microfluidics can be broadly defined as related to themanipulation of small fluidic volumes, typically those that are lessthan one milliliter. The concept that using microfluidic volumes inclinical assays would represent a significant improvement overconventional methods dates back to the late 1960's and early 1970's.While those working in the field appreciated that small volume analyseswould lead to portable instruments that required less laboratory space,improved precision and accuracy, increased throughput, reduced cost, andcompatibility with true automation, the systems developed at that timewere simple and did not realize the desired advantages of microfluidics.One early system was based on the analysis of 1-10 μl of sample and70-110 μl of reagents that were transferred and mixed in a smallcentrifugal rotor (Anderson, N. (1969). Computer interfaced fastanalyzers. Science 166: 317-24; and Burtis, C et al. (1972). Developmentof a miniature fast analyzer. Clinical Chemistry 18: 753-61). That rotoris likely the first reported microfluidic biochip.

In 1990, the theoretical foundation of the field of microfluidics wasfurther characterized by Manz, who coined the term “miniaturized totalchemical analysis system” or “μ-TAS”, to define what he consideredshould be the next generation of microfluidic devices. Manz proposedthat such devices should be capable of performing all required samplehandling steps. Manz' stated objectives helped establish the major goalin the modern era of microfluidics: to deliver a total, that is to say,fully integrated, analysis systems capable of performing a complexseries of process steps from the insertion of a sample to the generationof a result without operator intervention. It is the promise ofintegrating a complex series of sample manipulations and process stepsthat has led to the widespread adoption of the phrase “laboratory on achip.”

Work on centrifugal systems continued after the first early efforts andresulted in a commercial centrifugal system for sandwich immunoassaysthat used a microfluidic compact disc drive. The system was not fullyintegrated, however; and more accurately should be termed a workstationin which reagents were transferred to and from the compact discrobotically. Furthermore, the immunoassays were quite simple, based onpassing a sample through a capture column. Although the system usedmicrofluidic volumes to reduce reagent costs, it delivered neither onthe promise of microfluidics to provide a fully-integrated system nor onthe promise of microfluidics to integrate a complex series of samplemanipulations and process steps.

Similarly, other workers fabricated a spinning biochip in poly(methylmethacrylate) [PMMA] by soft embossing to fractionate plasma from wholeblood. There have been applications of spinning microfluidic biochips,for measurement of various analytes of interest in a blood sample.However, the devices have been developed for simple analytic tests, forexample establishing the amounts of analytes of interest in a bloodsample. The samples are not subjected to a complex series of samplemanipulations and process steps, and even after almost two decades ofdevelopment, can still have significant reliability problems correlatingwith established conventional assays.

In general, CD-based biochips have major drawbacks and limitations.First, they do not enable sufficient process complexity. These biochipsare limited to relatively simple processes such as certain cellseparations (serving as replacements for conventional centrifugationinstrumentation) and for high throughput immunoassays (serving asreplacements for the mixing and incubation of conventional assays).Second, the use of centrifugal force as the driver of fluidic transportis limiting. For example, the requirement to rotate the biochip placesprofound limitations on the approach to sample handling—either thesample must be introduced indirectly (requiring either manualintervention or additional instrumentation) or directly (e.g. a bloodcollection tube or swab would need to be subjected to centrifugation aswell). Furthermore, process flow in spinning biochips proceedsunidirectionally, and the radius of the biochip limits the areaavailable for sample process steps to take place (one of the factorsthat limits process complexity).

Concomitantly, alternatives to CD-based biochips have been studied. Anumber of groups have worked on microfluidic biochips based onmicrotiter plates using SBS (Society for Biomolecular Screening,Microplate Standards Development Committee, ANSI/SBS1, Danbury, Conn.,2004) standards. The SBS developed a series of published standards formicrotiter plates that include footprint (127.76 by 85.48 mm, 10,920.9mm²), height, and bottom outside flange dimensions and well positions.These standards have been incorporated by commercial manufacturers andacademic groups, regardless of the fabrication process utilized.Microfluidic microtiter plates have been developed for a number ofprocesses, including DNA purification and protein crystallization. Thesebiochips require sample pre-processing prior to introduction, performonly two steps of the DNA purification process, and do not perform anyanalysis of the DNA product of the process.

Similarly, other groups have used rectangular microfluidic plates basedon SBS standards for tasks such as the mixing of nanoliter volumereagents, medium exchange during live cell microscopy, and dispensingcells and reagents. Still other workers have adapted rectangularbiochips to be used in robotic systems for the high-throughputdispensing of reagents into wells. Another group developed a rectangularbiochip for protein detection by biological signal amplification in awell-in-a-well device.

In the progression of biochip development, a number of “integratedfluidic circuit” products based on the use of microfluidic features inan SBS format have been commercialized. For example, a biochip performssingle nucleotide polymorphism (SNP) genotyping using an array that caninterrogate 48 samples for each of 48 SNPs. The process is conducted byfirst preparing purified DNA and reaction mixes outside the microfluidicplate, placing the plate on a controller, priming the plate, loading andmixing reagents over 45 minutes on the plate, placing the plate on athermal cycler for PCR, and transferring the plate to a fluorescencedetection instrument. These biochips require sample pre-processing priorto introduction, require several manual steps during on-chip sampleprocessing, do not incorporate reagents, and do not perform any analysisof the DNA product of the process.

A similar commercial approach measures platelet adhesion using a wellplate microfluidic technology that integrates micron scale flow celldevices into SBS-standard well plates. The process requires a number ofpre-processing steps, including manually introducing a protein ofinterest to coat the microfluidic channels, perfusing the channels,washing, manually preparing the cell sample of interest, adding the cellsample to the microfluidic channels, and placing the plate onto aworkstation for further processing and analysis. A similar approach hasbeen applied to studying wound healing by exposing epithelial cells to avariety of compounds. These biochips require extensive samplepre-processing prior to introduction.

Much of the microfluidic biochip prior art is based on the use ofmaterials such as silicon or glass to fabricate biochips. However,silicon and glass biochips are prohibitively expensive to fabricate forhigh volume commercial applications (costing up to thousands of dollarsfor a single biochip) and are therefore impractical to be single-usedisposables. Moreover, the need to reuse these biochips leads toproblems with run-to-run contamination (an issue for human identity andclinical diagnostics), problems with instrumentation complexity if thebiochips are prepared for reuse in the instrument, and problems withlogistics if the biochips are prepared for reuse outside the instrument.

In order to realize the unfulfilled potential of microfluidics,microfluidic biochips and systems that are capable of performing acomplex series of processing steps for one or more samples in parallelin the setting of a fully-integrated, sample-in to results out system inwhich there is no requirement for operator manipulation is needed.However, the biochips developed to date perform only a subset of thesteps required for analysis, cannot perform complex series of processingsteps, and are not capable of producing analytical results on a singledevice. Furthermore, these biochips and systems use materials andmethods ill-suited to mass production.

SUMMARY OF THE TECHNOLOGY

In general, in one aspect, the technology described herein relates tobiochips having integration of all steps in a complex process from theinsertion of a sample to generation of a result, performed withoutoperator intervention. In an embodiment, a biochip in accordance withthe present technology includes microfluidic and macrofluidic featuresthat are acted upon by instrument subsystems in a series of processingsteps.

In another aspect, the technology relates to methods for fabrication ofcomplex biochips to include a high feature density. In one embodiment,the method includes injection molding of plastic materials to form thebiochip.

in another aspect, the technology relates to a biochip, which uponinsertion into an electrophoresis instrument having pneumatic, thermal,high voltage, and optical subsystems, and a process controller,generates a nucleic acid sequencing or sizing profile from at least onesample. The biochip includes: a macrofluidic processing subassembly inconnection with a fluidic subassembly and a pneumatic subassembly. Themacrofluidic processing subassembly includes at least one chamberadapted to receive a sample. The fluidic subassembly includes a fluidicplate, and at least one fluid transport channel and an amplificationchamber adapted to connecting to the thermal subsystem. The pneumaticsubassembly is adapted to connecting to the pneumatic subsystem of theinstrument, and to the subassemblies of the biochip. The pneumaticsubassembly includes a pneumatic plate and one or a plurality of drivelines to pneumatically drive fluids on instructions from said processcontroller. The biochip also includes a separation and detectionsubassembly adapted for connecting to the high voltage and opticalsubsystems and process controller on the instrument. The separation anddetection subassembly including a separation channel and a detectionregion positioned to send signals from each of the channels to theoptical subsystem on the instrument. The biochip is plastic, stationary,and unitary and a footprint of the fluidic and/or pneumatic plates isabout 86 mm by 128 mm or greater (e.g., is about 100×150 mm or greater,about 115×275 mm or greater, about 115×275 mm or greater, about 140×275or greater, 165×295 or greater.)

In another aspect, the technology relates to a biochip, which uponinsertion into an electrophoresis instrument having pneumatic, thermal,high voltage, and optical subsystems, and a process controller,generates a nucleic acid sequencing or sizing profile from at least onesample. The biochip includes: a macrofluidic processing subassembly inconnection with a fluidic subassembly and a pneumatic subassembly. Themacrofluidic processing subassembly includes at least one chamberadapted to receive a sample. The fluidic subassembly includes a fluidicplate, and at least one fluid transport channel and an amplificationchamber adapted to connecting to the thermal subsystem. The pneumaticsubassembly is adapted to connecting to the pneumatic subsystem of theinstrument, and to the subassemblies of the biochip. The pneumaticsubassembly includes a pneumatic plate and one or a plurality of drivelines to pneumatically drive fluids on instructions from said processcontroller. The biochip also includes a separation and detectionsubassembly adapted for connecting to the high voltage and opticalsubsystems and process controller on the instrument. The separation anddetection subassembly including a separation channel and a detectionregion positioned to send signals from each of the channels to theoptical subsystem on the instrument. The biochip is plastic, stationary,and unitary and a footprint of the fluidic and/or pneumatic plates isless than 10,920.0 mm² and does not conform to SBS standards

In another aspect, the technology relates to a biochip, which uponinsertion into an electrophoresis instrument having pneumatic, thermal,high voltage, and optical subsystems, and a process controller,generates a nucleic acid sequencing or sizing profile from at least onesample. The biochip includes a macrofluidic processing subassembly inconnection with a fluidic subassembly and a pneumatic subassembly. Themacrofluidic processing subassembly includes at least one macrofluidicfeature located therein or thereon and a chamber capable of receiving asample. The fluidic subassembly includes a fluidic plate, and at leastone feature located therein or thereon. The fluidic subassembly furthercomprising at least one fluid transport channel and an amplificationchamber adapted to connecting to the thermal subsystem. The pneumaticsubassembly is adapted to connecting to the pneumatic subsystem of theinstrument and to the subassemblies of the biochip. The pneumaticsubassembly includes a pneumatic plate and at least one feature locatedtherein or thereon. The pneumatic subassembly further includes one or aplurality of drive lines to pneumatically drive fluids on instructionsfrom said process controller. The biochip also includes a separation anddetection subassembly adapted for connecting to the high voltage andoptical subsystems and process controller on the instrument. Theseparation and detection subassembly includes at least one featurelocated therein or thereon. The separation and detection subassemblyfurther includes a separation channel and a detection region positionedto send signals from each of the channels to the optical subsystem onthe instrument. The biochip is plastic, stationary, and unitary, and aphysical state of the one or more features of the fluidic, pneumatic,and/or separation and detection subassemblies are adapted to change inscripted processes of 25 or more steps (e.g., 50 or more, 100 or more,200 or more.) In some embodiments, the scripted process steps result intwo or more resultant processing steps to occur within the biochip.

In another aspect, the technology relates to a unitary, stationarybiochip, which upon insertion into an electrophoresis instrument havingpneumatic, thermal, high voltage and optical subsystems, and a processcontroller, generates a nucleic acid sequencing or sizing profile fromat least one sample. The biochip includes a macrofluidic processingsubassembly in connection with a fluidic subassembly and a pneumaticsubassembly. The macrofluidic processing subassembly includes at leastone chamber adapted to receive a sample. The fluidic subassemblyincludes a fluidic plate having a top and bottom patterned thermoplasticsheet bonded thereon, to form at least one fluid transport channel oneach sides of the plate and an amplification chamber adapted toconnecting to the thermal subsystem. The pneumatic subassembly which isadapted to connecting to the pneumatic subsystem of the instrument, andto the subassemblies of said biochip. The pneumatic subassembly includesa pneumatic plate having a top patterned thermoplastic sheet bondedthereon, and one or a plurality of drive lines to pneumatically drivefluids on instructions from the process controller. The biochip alsoincludes a valve subassembly, positioned between and connected to thefluidic and pneumatic subassemblies and a separation and detectionsubassembly adapted for connecting to the high voltage and opticalsubsystems and process controller on the instrument. The separation anddetection subassembly including at least one separation channel, andfurther comprising a detection region positioned to send signals fromeach of the at least one separation channels to the optical subsystem onsaid instrument.

Some of the embodiments of this aspect of the technology include one ormore of the following features. One or more of the macrofluidic,pneumatic, fluidic, and separation and detection subsystems is formedfrom plastic. In some embodiments, the valve subassembly includes atleast one elastomeric valve. In some embodiments, the valve subassemblyincludes at least one non-elastomeric valve. In embodiments, theseparation and detection subassembly is oriented such thatelectrophoresis of the sample within the separation and detectionsubassembly is conducted in the opposite direction from a general flowof sample through the fluidic plate. In embodiments, the biochipincludes all reagents needed to process the at least one sample withinthe biochip.

In another aspect, the technology relates to a system for processing abiological sample. The system includes a biochip and a processcontroller. The biochip upon insertion into an electrophoresisinstrument having pneumatic, thermal, high voltage and opticalsubsystems, and the process controller, generates a nucleic acidsequencing or sizing profile from at least one sample. The biochipincludes a macrofluidic processing subassembly in connection with afluidic subassembly and a pneumatic subassembly. The macrofluidicprocessing subassembly includes at least one chamber adapted to receivea sample. The fluidic subassembly includes a fluidic plate having a topand bottom patterned thermoplastic sheet bonded thereon, to form atleast one fluid transport channel on each sides of the plate and anamplification chamber adapted to connecting to the thermal subsystem.The pneumatic subassembly which is adapted to connecting to thepneumatic subsystem of said instrument, and to the subassemblies of saidbiochip, includes a pneumatic plate having a top patterned thermoplasticsheet bonded thereon, and one or a plurality of drive lines topneumatically drive fluids on instructions from the process controller.The biochip also includes a valve subassembly positioned between andconnected to said fluidic and pneumatic subassemblies and a separationand detection subassembly adapted for connecting to the high voltage andoptical subsystems and process controller on the instrument. Theseparation and detection subassembly includes at least one separationchannel, and a detection region positioned to send signals from each ofthe at least one separation channels to the optical subsystem on theinstrument. The process controller of the instrument includesinstructions to perform 25 or more automated scripted process steps.

In another aspect, the technology features a method of manufacturing abiochip. The method includes injection molding a fluidic plate toinclude a fluidic top surface, a fluidic bottom surface and at least onethrough hole extending from the fluidic top surface to the fluidicbottom surface to connect the fluidic top surface to the bottom surface.Each of the fluidic top and bottom surfaces include a plurality ofmicrofluidic features. The method further includes injection molding apneumatic plate to include a pneumatic top surface, a pneumatic bottomsurface and at least one through hole extending from the pneumatic topsurface to the pneumatic bottom surface to connect the pneumatic topsurface to the bottom surface. Each of the pneumatic top and bottomsurfaces comprise a plurality of microfluidic features. The method alsoincludes aligning the pneumatic and fluidic plates to form the biochip,wherein a footprint of the fluidic and/or pneumatic plates is 86 mm by128 mm or greater, and at least one of the plurality of microfluidicfeatures of the fluidic plate and/or the pneumatic plate includes adraft angle of less than 2 degrees.

In another aspect, the technology features a method of performingsample-in to results out automated electrophoresis on at least oneunprocessed sample in a unitary biochip having a macrofluidic processingsubassembly in fluid communication with a fluidic subassembly and apneumatic subassembly. The method includes: (a) inserting the at leastone unprocessed sample into a chamber in the macrofluidic processingsubassembly; (b) inserting the biochip into an instrument having apneumatic subsystem, a thermal subsystem, a high voltage subsystem, anoptical system, and a process controller that can accept a predefinedprocess script and implement the process script automatically by readingthe process script and controlling the pneumatic subsystem, a thermalsubsystem, a high voltage subsystem, and optical system; (c) activatingthe process controller to carry out the process script, the processscript including instructions to: (i) initialize the biochip by applyingpressure from the pneumatic subsystem to valve lines of the pneumaticsubassembly or in fluid communication with the pneumatic subassembly;(ii) purify DNA from the unprocessed sample by applying pressure fromthe pneumatic subsystem to the macrofluidic processing subassembly torelease reagents stored therein, applying a pressure from the pneumaticsubsystem to the released reagents to form a cell lysate from theunprocessed sample, applying a pressure from the pneumatic subsystem topull the lysate through a purification filter in the fluidicssubassembly into a holding chamber to bind DNA in the lysate to thefilter, applying a pressure from the pneumatic subsystem to release awash solution stored in the macrofluidic processing subassembly to flowthrough the purification filter to remove contaminants from the boundDNA; applying a pressure to flow air through the purification filter todry the bound DNA, applying a pressure from the pneumatic subsystem torelease an elution solution stored in the macrofluidics processingsubassembly to release the bound DNA from the purification filter,generating an eluate containing purified DNA; (iii) transport the eluateto a reconstitution chamber containing a lyophilized PCR reaction mix byapplying pressure from the pneumatic subsystem to the pneumaticsubassembly; (iv) transport the reconstituted PCR reaction mix to athermal cycling chamber in the fluidics subassembly; (v) apply heat tothe thermal cycling chamber by initiating a thermal cycle protocol togenerate labeled amplicons from the PCR mixture; (vi) transport thelabeled amplicons and a formamide reagent stored in the macrofluidicprocessing subassembly to a joining chamber in the fluidics subassemblyby applying pressure from the pneumatics subsystem to the pneumaticsubassembly; the labeled amplicons and formamide reagent forming aformamide-PCR product mixture; (vii) transport the formamide-PCR productmixture by applying a pressure from the pneumatic system to thepneumatic subassembly to flow the mixture to an ILS cake chamber formixing with an ILS cake to generate a separation and detection sample;(viii) transport the separation and detection sample into a cathodechamber within the fluidics subassembly by applying a pressure from thepneumatic subsystem to the pneumatic subassembly; (ix) prepare cathodeand anode of the fluidics subassembly for separation of the separationand detection by applying a voltage from the high voltage subsystem tobias the cathode and anode; (x) transport gel from a gel reagentreservoir in the fluidics subassembly to a cathode chamber and wastechamber of the fluidics subassembly by application of pressure from thepneumatics subsystem to the pneumatic subassembly; (xi) inject andseparate the separation and detection sample in the separation channelby applying a voltage through the high voltage subsystem to a cathodeand anode; and (xii) effect fluorescence of separated components of theseparation and detection sample by activating a laser in the opticalsubsystem. The method also includes automatically detecting afluorescent signal of the separated components to provide separation anddetection sample results from at least one unprocessed samples.

In another aspect, the technology relates to a system for providing atleast two types of sample-in to results-out processing for at least onebiological sample. The system includes a stationary biochip and aninstrument including pneumatic, thermal, high voltage and opticalsubsystems for interfacing with the biochip, and a process controller.The stationary biochip includes a macrofluidic processing subassembly inconnection with a fluidic subassembly and a pneumatic subassembly. Themacrofluidic processing subassembly including at least one chamberadapted to receive a sample. The fluidic subassembly includes a fluidicplate, and at least one fluid transport channel and an amplificationchamber adapted to connecting to the thermal subsystem. The pneumaticsubassembly which is adapted to connecting to the pneumatic subsystem ofsaid instrument, and to the subassemblies of said biochip, includes apneumatic plate and one or a plurality of drive lines to pneumaticallydrive fluids on instructions from the process controller. The biochipalso includes a separation and detection subassembly adapted forconnecting to the high voltage and optical subsystems and processcontroller on the instrument. The separation and detection subassemblyincluding a separation channel, and a detection region positioned tosend signals from each of the channels to the optical subsystem on theinstrument. The biochip further includes at least two distinct pathwaysfor sample processing. Each of the at least two distinct pathways isdedicated to different analytical processes. The process controller ofthe instrument includes a set of instructions to process the at leastone biological sample in the different analytical processes. In someembodiments, the different analytical processes are STR analysis andsingle nucleotide polymorphism analysis. In some embodiments, thedifferent analytical processes are multiplexed amplification and DNAsequencing. In some embodiments, the different analytical processes areSTR analysis and mitochondrial DNA sequencing. In some embodiments, thedifferent analytical processes are reverse transcription PCR andconventional PCR. In some embodiments, the different analyticalprocesses are DNA sequencing and single nucleotide polymorphismanalysis.

In another aspect, the technology relates to a reagent storage containerfor a biochip including a macrofluidic block and cover. The macrofluidicblock includes a reagent storage chamber having a top end and a bottomend; a first foil seal bonded to the bottom end; and a second foil sealbonded to the top end. A reagent is stored in the foil-sealed chamberand is released by the application of pneumatic pressure through thecover such that the top and bottom foils burst, releasing the contentsof the reagent storage chamber.

Embodiments of the above aspect can include one or more of the followingfeatures. In some embodiments, the reagent storage container isconnected to a fluidic subassembly. In some embodiments, the reagentstorage container further includes a spacer plate placed between thecontainer and the fluidic subassembly such that the spacer plate issized to accommodate expansion of the first foil prior to bursting.

There are numerous advantages of the present technology including, butnot limited to, minimal to no operator intervention and reducedfabrication costs.

DESCRIPTION OF THE DRAWINGS

The advantages of the technology described above, together with furtheradvantages, may be better understood by referring to the followingdescription taken in conjunction with the accompanying drawings. Thedrawings are not necessarily to scale, emphasis instead generally beingplaced upon illustrating the principles of the technology.

FIG. 1 is a side view schematic of an embodiment of a single tubereagent storage and release device.

FIG. 2 is a photograph of an embodiment of a spacer plate included in a5-sample biochipset embodiment utilizing reagent storage releasebursting.

FIG. 3 is a cross-section view schematic of a portion of a spacer plate.

FIG. 4 is a graph of burst pressure versus chamber diameter for aluminumfoils of various thicknesses.

FIG. 5 is a perspective view schematic of a reagent storage release unitincluding six chambers of reagent storage.

FIG. 6 is top perspective view of the reagent storage release unit ofFIG. 5 showing a top foil bonded to a top end of each chamber.

FIG. 7 is a top view illustration of two foils of a reagent storagerelease unit after bursting.

FIG. 8 is a top view schematic of an embodiment of a pneumaticallyactuated elastomeric valve structure.

FIG. 9 is a cross-sectional view schematic of the pneumatically actuatedvalve structure of FIG. 8.

FIG. 10 is a top view schematic of an embodiment of an elastomericmembrane valve with PSA tape.

FIG. 11 is a cross-sectional view schematic of the valve of claim 10.

FIG. 12 is a top view schematic of an embodiment of a pneumaticallyactuated valve with a rigid valve membrane.

FIG. 13 is a cross-sectional view of the pneumatically actuated valvewith rigid valve membrane of FIG. 12.

FIG. 14 is a top view schematic of an embodiment of a clampedelastomeric membrane valve.

FIG. 15 is a cross-sectional view of the clamped elastomeric membranevalve of FIG. 12.

FIG. 16 is a cross-sectional view schematic of a vent membraneconfiguration used in an embodiment of a biochip in accordance with thepresent technology.

FIG. 17 is a cross-sectional view schematic of a vent membraneconfiguration used in another embodiment of a biochip in accordance withthe present technology.

FIG. 18 is a top view schematic of a pneumatic plate with the manifoldand surrounding areas expanded of an embodiment of a biochip inaccordance with the present technology.

FIG. 19 is a bottomview schematic of a pneumatic plate with the manifoldand surrounding areas expanded of an embodiment of a biochip inaccordance with the present technology.

FIG. 20 is a transparent view schematic of a fluidic plate with thepurification region and surrounding areas expanded of an embodiment of abiochip in accordance with the present technology.

FIG. 21 is a photograph of a pneumatically activated cylinder used toexert a controlled force onto a biochip.

FIG. 22 is a bar graph comparing signal strength at each STR locus forvarious pressures applied to the biochip by the pneumatically activatedcylinder of FIG. 21. In the graph, pressures of 75, 112, 187, 224, 299,and 336 psig were applied, and, for each locus, signal strengths arerecorded for each pressure (increasing pressures from left to right).

FIG. 23 is a top view schematic of an embodiment of an injection moldedbiochip in accordance with the present technology.

FIG. 24 is a top view schematic of an embodiment of fluidic layer 1 of abiochip in accordance with the present technology.

FIG. 25 is a top view schematic of an embodiment of a fluidic layer 2 ofa biochip in accordance with the present technology.

FIG. 26 is a top view schematic of an embodiment of a pneumatics layer 1of a biochip in accordance with the present technology.

FIG. 27 is a top view schematic of an embodiment of a pneumatic later 2of a biochip in accordance with the present technology.

FIG. 28 is a transparent top view schematic of an embodiment of afluidics subassembly including the first fluidic plate of FIG. 24 bondedto the second fluidic plate of FIG. 25.

FIG. 29 is a transparent top view schematic of an embodiment of apneumatic subassembly including the first pneumatic plate of FIG. 26bonded to the second pneumatic plate of FIG. 27.

FIG. 30 is a transparent top view schematic of a biochip formed by theattachment of the pneumatics subassembly of FIG. 29 to the fluidicssubassembly of FIG. 28.

FIG. 31 is a photograph of an embodiment of a pneumatic and thermalcycling instrument for use in connection with biochips.

FIG. 32 is a STR profile for a PCR analysis of a sample within a biochipin accordance with the present technology.

FIG. 33 is a top view schematic of an embodiment of a fluidic plateincluded in an embodiment of a fluidic assembly in accordance with thepresent technology.

FIG. 34 is a bottom view schematic of the fluidic plate of FIG. 33.

FIG. 35 is a transparent view schematic of the fluidic plate showing thefeatures of both the top and bottom sides of the plate.

FIG. 36 is a top view schematic of an embodiment of a patterned thinfilm for attachment to the top of the fluidic plate.

FIG. 37 is a bottom view schematic of an embodiment of a patterned thinfilm for attachment to the bottom of the fluidic plate.

FIG. 38 is a top view schematic of an embodiment of a fluidic plate witha line illustrating a path a single sample takes through the fluidicplate.

FIG. 39 is a top expanded view schematic of the fluidic plate of FIG. 38showing a portion of the path through a purification region.

FIG. 40 is a top expanded view schematic of the fluidic plate of FIG. 38showing a portion of the path through an amplification region.

FIG. 41 is a top expanded view schematic of the fluidic plate of FIG. 38showing a portion of the path through a separation and detection region.

FIG. 42 is a top view schematic of an embodiment of a pneumatic plateincluded in an embodiment of a pneumatic assembly in accordance with thepresent technology.

FIG. 43 is a bottom view schematic of the pneumatic plate of FIG. 42.

FIG. 44 is a transparent view of the pneumatic plate of FIG. 42 showingthe features of both top and bottom sides of the pneumatic plate.

FIG. 45 is a top view schematic of an embodiment of a patterned thinfilm for attachment to the top side of the pneumatic plate.

FIG. 46 is a bottom view schematic of an embodiment of a patterned thinfilm for attachment to the bottom side of the pneumatic plate.

FIG. 47 is a transparent view schematic of an embodiment of macrofluidicblock included in an embodiment of a macrofluidic processingsubassembly.

FIG. 48 is a top view schematic of an embodiment of a top layer of acover to the macrofluidic block.

FIG. 49 is a bottom view schematic of an embodiment of a top layer ofthe cover.

FIG. 50 is a top transparent view schematic of the top layer of thecover.

FIG. 51 is a top view schematic of another embodiment of a second layerof a cover to the macrofluidic block.

FIG. 52 is a top view schematic of another embodiment of a third layerof a cover to the macrofluidic block.

FIG. 53 is a bottom view schematic of an embodiment of a third layer tothe cover of FIG. 52.

FIG. 54 is a transparent top view schematic of a third layer to thecover.

FIG. 55 is a top view schematic of an embodiment of a separation anddetection biochip for pressure injection.

FIG. 56 is a top view schematic of an embodiment of a separation anddetection biochip for cross injection for electrokinetic transport of asample.

FIG. 57 is a side view schematic stack up of an embodiment of a biochipin accordance with the present technology.

FIG. 58 is a photograph of an embodiment of a biochip assembly inaccordance with the present technology and including arrows to indicatethe direction of process flows.

FIG. 59 is a photograph of the biochip assembly of FIG. 58. The biochipassembly interfaces with an optical subsystem, a thermal subsystem, ahigh voltage subsystem and pneumatic subsystem, and process controlsubsystem.

FIG. 60 is a control electropherogram generated for the control ILSsample (fragments fluorescently labeled with ROX) within an embodimentof a biochip in accordance with the present technology.

FIG. 61 is an electropherogram showing the sizes of the STR fragmentsgenerated for one buccal swab sample following an automated script. Eachof fluorescent dyes used to label the STR primers in the PCR reactionmix and the ILS are shown in individual panels. Fluorescein-labeledfragments are shown in the top panel, JOE-labeled fragments are shown inthe second panel from the top, TAMRA-labeled fragments are shown in thethird panel from the top, and ROX-labeled ILS fragments are shown in thebottom panel.

FIG. 62 is a top view schematic of an embodiment of a fluidic plate of afluidic subassembly in accordance with the present technology.

FIG. 63 is a bottom view schematic of the fluidic plate of FIG. 62.

FIG. 64 is a transparent view schematic of the fluidic plate showingboth top side features of FIG. 62 and bottom side features of FIG. 63.

FIG. 65 is a photograph of an embodiment of an injection molded fluidicplate.

FIG. 66 is a top view schematic of an embodiment of a top patterned thinfilm for attachment to the top side of the fluidic plate of FIG. 65.

FIG. 67 is a bottom view schematic of an embodiment of a bottompatterned thin film for attachment to the bottom side of the fluidicplate of FIG. 65.

FIG. 68 is a top view schematic of an embodiment of a fluidic plate witha line illustrating a path of a single sample takes through the fluidicplate.

FIG. 69 is a top expanded view schematic of the fluidic plate of FIG. 68showing a portion of the path through a purification region.

FIG. 70 is a top expanded view schematic of the fluidic plate of FIG. 68showing a portion of the path through a PCR section.

FIG. 71 is a top expanded view schematic of the fluidic plate of FIG. 68showing a portion of the path through a separation and detection region.

FIG. 72 is a top view schematic of an embodiment of a pneumatic plate ofa pneumatic assembly in accordance with the present technology.

FIG. 73 is a bottom view schematic of the pneumatic plate of FIG. 72.

FIG. 74 is a transparent top view of the pneumatic plate showing bothtop and bottom sides of the pneumatic plate of FIGS. 72 and 73.

FIG. 75 is a photograph of an embodiment of an injection moldedpneumatic plate.

FIG. 76 is a top view schematic of an embodiment of a patterned thinfilm for attachment to a top side of the injection molded pneumaticplate of FIG. 75.

FIG. 77 is a table representing a relationship of scripted processingsteps and resulting processing steps for a portion of an automatedprocess for use on a biochip.

FIG. 78 is a flowchart of an embodiment of a sample splitting anddilution microfluidic circuit.

FIG. 79 is a graph showing background fluorescence of several materialsat different thicknesses.

FIG. 80 is a table summarizing results of fluorescence data collectedwith and without a notch filter.

FIG. 81 is a photograph of a second macrofluidic processing assemblytermed the Matrix loading assembly.

FIG. 82 is a photograph of a biochipset fabricated by CNC-machining

FIG. 83 is an electropherogram generated for the allelic ladder sample(fragments fluorescently labeled with fluorescein, JOE, TAMRA, and ROX)of Example 10.

FIG. 84 is an electropherogram depicting single base pair resolution, asindicated by the clear separation of the Th01 9.3 and 10 alleles shownwithin a portion of the allelic ladder from one of the buccal swabsamples of FIG. 85.

FIG. 85 is electrophertic data generated using the biochip of Example10.

FIG. 86 is a photograph of an embodiment of an injection molded biochip.

FIG. 87 is a photograph showing the top view of the injection moldedbiochip of FIG. 86.

FIG. 88 is a photograph showing the bottom view of the injection moldedbiochip of FIG. 86.

FIG. 89 is a raw data plot of signal strength (RFU) versus S&D run timefor the biochip of Example 11.

DETAILED DESCRIPTION OF THE INVENTION

The biochips described herein achieve the fundamental goal of the fieldof microfluidics: the integration of all steps in a complex process,from the insertion of a sample to the generation of a result, performedin a single instrument without operator intervention. In one aspect, wepresent herein novel biochips that are fully integrated and capable ofperforming complex sample in to results out analyses including celllysis, DNA purification, multiplexed amplification, and electrophoreticseparation and detection to generate short tandem repeat (STR) profilesfrom forensic samples; cell lysis, DNA purification, multiplexedamplification, Sanger sequencing, ultrafiltration, and electrophoreticseparation and detection to generate DNA sequence from clinical samples;nucleic acid purification, reverse transcription, multiplexedamplification, Sanger sequencing, ultrafiltration, and electrophoreticseparation and detection to generate DNA sequence from biothreatsamples, and nucleic acid purification, library construction, and singlemolecule sequencing to generate genomic DNA sequences from human,bacterial, and viral clinical and research samples.

Sample manipulations that can be performed in the biochips of theinvention include combinations of nucleic acid extraction; cell lysis;cell separation; differential cell lysis; differential filtration; totalnucleic acid purification; DNA purification; RNA purification; mRNApurification; protein purification; pre-nucleic acid amplificationcleanup; nucleic acid amplification (e.g. both singleplex and multiplexend-point PCR, Real-time PCR, reverse transcription PCR, asymmetric PCR,nested PCR, LATE PCR, touchdown PCR, digital PCR, rolling circleamplification, strand displacement amplification, and multipledisplacement amplification); Y-STR amplification; mini-STRamplification; single nucleotide polymorphism analysis; VNTR analysis;RFLP analysis; post-nucleic acid amplification cleanup; pre-nucleic acidsequencing cleanup; nucleic acid sequencing (e.g. Sanger sequencing,pyrosequencing, and single molecule sequencing); post-nucleic acidsequencing cleanup; reverse transcription; pre-reverse transcriptioncleanup; post-reverse transcription cleanup; nucleic acid ligation; SNPanalysis; nucleic acid hybridization; electrophoretic separation anddetection; immunoassays; binding assays; protein assays; enzymaticassays; mass spectroscopy; and nucleic acid and protein quantification.

When characterizing the structure and functions of a biochip, at leasttwo classes of complexity can be considered. Sample number complexityrefers to the number of independent samples that are processed on thebiochip. Process complexity refers to the number of sequentialmanipulations to which each sample (and sample byproducts) is subjectedon the biochip. The biochips of the invention have high levels of bothsample number and process complexities.

In another aspect, the novel biochips of the invention integrate a largenumber of processing steps in order to achieve a fully integrated,sample-in to results-out system. We define two categories of biochipprocessing steps to express the complexity of these biochips. Scriptedprocessing steps are the actions performed as a result of automated,computer-controlled scripts that cause a direct action on a featurewithin or on the biochip and involve various subsystems of theinstrument that interface with the biochip. The actions cause a changeto the physical state of a feature in the biochip (e.g. a valve membraneis moved to close a valve) or to a sample or air within the biochip(e.g. a fluorescent dye in a sample is excited, air is passed through apneumatic drive). In addition, the action can cause a change to thephysical state of a feature in the biochip and a sample within thebiochip simultaneously (e.g. when the thermal chambers are heated, thesample within them are also heated).

The instrument subsystems perform the scripted actions and include thepneumatic subsystem, which applies pressure to burst foils, increase andreduce pressure on valves, and push liquids, gasses, and solids; thehigh voltage subsystem, which applies an electrical current toelectrophorese macromolecules; the optical subsystem, which applieslight to excite and detect a sample in electrophoresis, quantitation,amplification, immunoassays, and chemical assays; and the thermalsubsystem, which applies heat for cell lysis, nucleic acid denaturation,electrophoretic uniformity, thermal cycling, and cycle sequencing. Thatis, every scripted processing step is a specific instruction of anautomated script.

The scripted process steps define direct actions that take place onfeatures within or on the biochip via an interface with a subsystem ofthe instrument. The biochip features can be microfluidic, macrofluidic,or a combination of both, and the script acts upon these features atdefined locations in a defined sequence. For example, the actuation of agiven valve on the biochip is considered a single scripted process step,even if several changes to the instrument are required as prerequisitesto effect that single step (e.g. activating a pump and drive line toallow the valve to be closed).

In quantifying the number of scripted process steps, directly repetitivesteps such as those in PCR amplification (e.g. many directly repeatingcycles of denaturation, annealing, and extension, each characterized bya change in temperature) are considered as the number of steps in asingle cycle. That is to say, a PCR amplification reaction that consistsof Hotstart 93° C. for 20 seconds (1 step) followed by 31 cycles of [93°C. for 4 seconds, 56° C. for 15 seconds, and 70° C. for 7 seconds] (3steps) followed by a final extension of 70° C. for 90 seconds (1 step)represents a total of 5 scripted processing steps. One feature can beacted upon during one or more scripted process steps. In this case, eachindependent act counts as one step; for example, if a given valve isactuated seven times during a process, this would count as 7 steps.Several features can be acted upon in parallel during the process of anindividual sample. For example, if three valves are actuated while achamber of the biochip is heated, this would count as four scriptedprocessing steps (in other words, four new actions performed on featureson or within the biochip). Finally, once raw data is generated from theprocess, any data processing (e.g. color correction) or data analysis(e.g. allele or base calling) are not scripted processing steps. Thebiochips of the current invention perform 25, 50, 75, 100, 125, 150,175, 200, 250, 300, 400, 500, 750, 1000, and greater than 1000 scriptedprocessing steps.

Resultant processing steps are the effects of scripted processing stepsand include the movement of the liquid sample throughout the biochipthrough channels, chambers, and filter and membrane features; the dryingof filters and membranes, the thermal cycling of reaction mixes; theresuspension of lyophilized reagents, the joining of various liquids,the mixing of liquids, the homogenization of liquids, theelectrophoretic transport of sample macromolecules through variousmatrices, and the excitation of those macromolecules. It follows thatthe number of scripted processing steps will generally be greater than(and never be less than) the resultant processing steps. For example, itmay be necessary to close several valves and change several drivelinepneumatic pressures (multiple scripted processing steps) to cause themovement of a fluid plug from a given channel to an adjacent chamber (asingle resultant processing step). Example 6 and FIG. 77 describe therelationship between scripted and resultant processing steps for aportion of an automated scripted. The biochips of the current inventionperform 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,125, 150, 175, 200, 250, and greater than 250 resultant process steps.

Total processing steps is the sum of the scripted and resultantprocessing steps. The greater the number of total processing steps, thegreater the process complexity. The biochips of the current inventionperform 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, 500, 750,1000, 1500, 2000, and greater than 2000 total processing steps.Scripted, resultant, and total processing steps refer to the processingof a single sample. If multiple samples are processed in parallel on agiven biochip, the total number of processing steps is, by convention,the same as if only one sample is processed. Similarly, if multiplesamples are processed identically in series (e.g. if a first samplepasses through the biochip and then a second sample follows behind it,undergoing the same process), the total number of processing steps isthe same as if only single sample is processed. The importance of thesedistinctions is that the complexity biochips of the invention is basedon the intricate series of manipulations of a single sample as opposedto biochips that perform relatively small numbers of process steps butrepeat them on small or large numbers of samples.

The biochips of the invention allow integration of two or more of thefunctions noted above. Accordingly, a limitless number of combinationscan be designed into the biochip, allowing a complex set ofmanipulations to be completed on the biochip. Process complexity willinvolve a number of manipulations based on the microfluidic elementsnoted above. For example, the biochips of the invention accepting asample, potentiate mixing of lysis reagents, lyse the sample by chaoticbubbling, filter the lysate, meter the lysate, bind the lysate to asilica membrane, washing the membrane, elute nucleic acids from themembrane, homogenize the eluate to yield a purified DNA solution, meterthe DNA solution, reconstitute a lyophilized PCR reaction mix to yield aPCR reaction, mix and homogenize the reaction mix, meter the reactionmix, conduct rapid multiplexed amplification, meter a portion of thecompleted PCR reaction, reconstitute a sizing standard with the meteredPCR reaction to yield a solution for electrophoresis, mix and homogenizethe solution, combine the solution with reagents for electrophoresis,inject the material into an electrophoretic channel, conductelectrophoretic separation, detect the separated DNA fragments based onfluorescence, generate an electropherogram based on the raw fluorescencedata, color correct the raw data, identify the PCR peaks in theprocessed data, and analyze the called peaks (e.g. to generate an STRprofile for human forensic identification, to generate a multiplexedprofile for diagnosing an infectious disease, or to generate amultiplexed profile to identify a biothreat agent). One skilled in theart will appreciate that the biochips of the invention can be designedto perform a multitude of different types of analysis with essentiallylimitless process complexity.

Overview of Biochip Design

The biochips of the invention are unitary and represent a single,solitary structure. These unitary biochips are comprised of manycomponent parts, but all the parts, whether microfluidic ormacrofluidic, are directly and permanently attached, without the use oftubing to carry liquid, solids, or gasses from one portion of thebiochip to another. An advantage of unitary biochips is that it can beplaced into the corresponding instrument as a unit, requiring notechnical skill on the part of the operator. Furthermore, the absence ofconnecting tubes enhances robustness by minimizing leakage.

Preferably, the major components of the unitary biochips of theinvention are plastics. Glass, quartz, and silicon wafers are absent orminimized in the inventive biochips as they are quite costly tofabricate. It is preferable that all components (with the exception ofinserted elements such as reagents, filters, membranes, metal foils,electrode pins, and electrode strips and assembly materials such asgaskets, pressure sensitive adhesives (PSA) and tapes) are made ofplastic. It is also preferred that each sample pathway in the biochip issingle use; this prevents run-to-run contamination, simplifies design,and enhances ease of use. Accordingly, in one aspect, this inventionprovides single use, disposable plastic biochips.

We define two broad classes of microfluidic biochips based on the drivemechanisms used to transfer solutions within them. Centrifugalmicrofluidic biochips have circular footprints and rotate to providecentrifugal force to drive fluids from location to location within thebiochip. Stationary (or non-centrifugal) microfluidic biochips have awide range of footprints (including rectangular orsubstantially-rectangular footprints) and are characterized by drivesthat are not provided by rotation of the biochip.

Biochips of this invention are stationary, meaning that they do notrequire rotation to generate centrifugal force to drive fluidsthroughout the biochip (although the biochips can be subjected tomovement in general and translational movement in particular). Instead,the biochips of the inventions have drive mechanisms that includepneumatic, mechanical, magnetic, and fluidic. A variety of methods canbe used for liquid transport and controlled liquid flow. One method ispositive-displacement pumping, in that a plunger in contact with eitherthe liquid or an interposing gas drives the liquid a precise distancebased on the volume displaced by the plunger during the motion. Anexample of such a method is a syringe pump. Another method is the use ofintegrated elastomeric membranes that are pneumatically, magnetically,or otherwise actuated. A preferred method for driving fluids andcontrolling flow rates is to use a pneumatic drive to apply vacuum orpressure directly on the liquids themselves, by altering the pressure atthe leading, trailing, or both menisci of the liquids. Appropriatepressures (typically in the range of 0.05-300 psig and usually in therange of 0.5-30 psig) are applied to achieve desired flowcharacteristics. Flow can also be controlled by selecting appropriatesizing of the fluidic channels, as the flow rate is proportional to thepressure differential across the fluid and the hydraulic diameter to thefourth power and inversely proportional to the length of the channel orthe liquid plug and the viscosity. Furthermore, flow can be controlledand parallel samples aligned by incorporating vent membranes along theflow channel.

The biochips of the invention function with a single instrument to carryout a complex chemical analysis. The inventive biochips described hereinimprove processing efficiency and data quality by eliminating sampletransfer from instrument to instrument, improving ease of use byminimizing or eliminating operator requirements (a single machine may,using an appropriate script and user interface), eliminate any need foroperator actions, improving the ability to ruggedize the system (allruggedization subsystems can be incorporating into a single instrument),and reducing the cost of the instrumentation required as well as thelaboratory infrastructure required to process samples. In fact, in oneembodiment, having the biochip function in a single instrument canobviate the need for a laboratory environment.

The instrument provides all the subsystems required for the completionof sample processing. These subsystems include high and low voltagepower subsystems, thermal cycling subsystems, pneumatic subsystems,magnetic subsystems, mechanical subsystems, ultrasonic subsystems,optical subsystems, ruggedization subsystems, process controlsubsystems, and computer subsystems. The instrument to biochip interfacemay involve one or more of these subsystems, depending on themicrofluidic drive and the series of processes to be performed withinthe biochip. In the case of the examples herein, the interface of thebiochips and the instrument are pneumatic, electrical, optical, andmechanical. The size of the instrument is determined by subsystemdimensions, biochip dimensions, and throughput. The instruments mayweigh approximately 10 pounds, 25 pounds, 50 pounds, 100 pounds, 150pounds, 200 pounds, or greater than 200 pounds. Similarly, the volume ofthe instrument may be 0.5 cubic feet, or 1, 2, 4, 6, 10, 15, 20 orgreater than 20 cubic feet.

Furthermore, the biochip and instrumentation of the invention aredesigned to be operable outside of conventional laboratory environments.Depending upon the application, they can be ruggedized to withstandtransport and extremes of temperature, humidity, and airborneparticulates. Use of the invention by non-technical operators inoffices, out of doors, in the battlefield, in airports, at borders andports, and at the point-of-care will allow much broader application ofgenetic technology in society. The use of unprocessed samples furthersupports the broad application of the teachings of the invention.

The inventive microfluidic systems, instruments and biochips set forthherein can be single- or multi-use. The advantages of single-usedisposable biochips include:

-   -   Minimization of cross contamination. In a multi-use device, the        sample or a component of the sample may be present in the device        following washing; the remnant may contaminate a subsequent        analysis. Single-use disposable devices are inherently free of        remnant sample. This is achieved, in part, by the novel sample        processing channels and methods of manufacturing them, in which        there is no sample-to-sample channel communication.    -   Minimization of technical requirements. A multi-use device        requires equipment to clean a spent device and prepare it for        subsequent reuse. The single-use disposables of this invention        are not burdened by these requirements.    -   Minimization of operator requirements. In addition to the skill        required to carefully clean and prepare a spent device for        subsequent use, reagents must be reloaded into a multiuse device        either by insertion into the device or the instrument. By        incorporating all reagents into a single-use disposable        microfluidic device, the operator need not manipulate or be        exposed to reagents at all. The operator need only insert the        sample into the device and press a start button. It follows that        the inventive biochips described herein can be operated by        unskilled or minimally trained operators and may be performed        outside the typical laboratory environment. Analysis of samples        at the point-of-care, the battlefield, military checkpoints,        embassies, sites critical to national security, and developing        regions of the world is possible with these inventive biochips.        Autonomous collection and analysis of samples as applied to        applications including biothreat detection also benefit from a        minimization of operator requirements. Work describing an        approach that essentially eliminates sample handling        requirements, further simplifying and broadening the        possibilities for analytic methodologies is described in U.S.        patent application Ser. No. 12/699,564, filed Feb. 3, 2010,        entitled “Nucleic Acid Purification,” which is hereby        incorporated by reference in its entirety.    -   Increase in System Throughput. The minimization of technical and        operator requirements allows an increase of the number of        samples analyzed per unit time. The dense packing of features        allows for processing multiple samples in parallel, and the use        of large biochips allows further increase in throughput.        Furthermore, the reversal of the direction of process flow (e.g.        electrophoresis vis a vis purification and amplification) allows        for a smaller, more compact biochip and instrument.    -   Minimization of Cost. Minimization of labor requirements and        elimination of overhead reduce costs.

The inventive biochips described herein may have dimensions greater thanthat of the SBS standard microtiter plate. The dimensions of the biochipfootprint are 86 by 128 or greater, 100×150 mm or greater, 115×275, mmor greater, 140×275 or greater, and 165×295 or greater. Also theinventive biochips encompass footprints of less than 10,920.0 mm² thatdo not conform to SBS standards. The teachings of the invention allowthe design and fabrication of biochips with an unprecedented degree ofsample number complexity and process complexity. There is no limit tothe possible dimensions of the biochip footprint. The footprint can bemade in accordance to the needs of a particular user and requirements ofa given application.

As the sample number and process complexities of the biochips of theinvention increase, the feature density of the biochips increases. Inthis context, microfluidic features are defined as any area of the layerin question that is either cut into by CNC-machining or molded around byinjection molding. Feature density is the ratio of the number ofmicrofluidic features to the surface area of the layer in question. Forexample, the biochips of Example 5 and 6 have the following densities:the top and bottom of the fluidics layer have approximately 20.2% and6.6% respectively of their surface areas occupied by microfluidicfeatures. The top and bottom of the pneumatics layer have 7.5% and 8.8%of their surface areas occupied by microfluidic features. The 16-sampleversion of the same biochip occupies essentially the same footprint yethas even higher densities: 19% and 13.7% for fluidics and 18.3% and 25%for pneumatics. Local densities on the layers can be 60% or higher(certain regions of the biochips of Examples 4, 5, and 6 have localdensities of over 64% in regions of approximately 3 cm². The pneumaticsand fluidic biochips of the invention have 5, 10, 15, 20, 25, 30, 40,50, 60, 70, 80, and greater than 80% of their surface areas occupied bymicrofluidic features. Macrofluidic features also contribute to density.

Features of the Biochips of the Invention

The biochips of the invention comprise thermoplastic layers that containmicrofluidic features such as one or more fluid transport channels(which may be independent, connected, or networked), through holes,alignment features, liquid and lyophilized reagent storage chambers,reagent release chambers, pumps, metering chambers, lyophilized cakereconstitution chambers, ultrasonic chambers, joining and mixingchambers, mixing elements, membrane regions, filtration regions, ventingelements, heating elements, magnetic elements, reaction chambers, wastechambers, membrane regions, thermal transfer regions, anodes, cathodes,and detection regions, drives, valve lines, valve structures, assemblyfeatures, instrument interface regions, optical windows, thermalwindows, and detection regions. The biochips may also containmacrofluidic features, that is, features that are larger versions of thesame types as the microfluidic features listed above. For example, thebiochip may contain a microfluidic reagent storage chamber that fold 30microliters of liquid and a macrofluidic reagent storage chamber thatholds five milliliters of liquid. The incorporation of both microfluidicand macrofluidic features on the biochips of the invention allow a givenbiochip to meet both macrofluidic and microfluidic processingrequirements. This is an advantage when samples requiring relativelylarge volumes (such as a forensic swab or a clinical blood sample) areto be processed at a microfluidic scale.

The biochips may also incorporate thin films including patterned thinfilms, spacer layers, adhesive layers, elastic and non-elastic layers,and detection regions. The biochips may also incorporate layers withfeatures based on particular drive mechanisms (e.g. actuation lines forpneumatic, mechanical, magnetic, and fluidic drives). Note that themicrofluidic biochips may also contain macrofluidic regions,particularly for processing forensic, clinical diagnostic, and biothreatsamples.

The biochips of the invention allow nucleic acids and other biologicalcomponents from unprocessed biological samples to be purified,manipulated, and analyzed. Unprocessed biological samples are those thatare collected by an individual and then inserted into the samplereceiving chamber of the biochip with no intermediate processing steps(although the sample collection device may be labeled and/or storedprior to processing). The operator need only collect or otherwise obtainthe sample, insert the sample into the apparatus, insert the apparatusinto the instrument (not necessary if the apparatus was previouslyplaced in the instrument), and press a start button. No processing,manipulation, or modification of the sample is required prior toinsertion in the apparatus—the operator does not have to cut a swab,open a blood tube, collect a tissues or biologic fluid, transfer asample to another holder, or expose the sample to a reagent or acondition (e.g. heat, cold, vibration). Accordingly, the operator neednot have extensive training in the biological sciences or laboratorytechniques. Optionally, the biochips of the invention can acceptprocessed biological samples (e.g. a cell lysate for subsequentpurification), but such applications may require an operator withtechnical training.

In practice, biological samples are collected using a myriad ofcollection devices, all of which can be used with the biochips of theinvention. The collection devices will generally be commerciallyavailable but can also be specifically designed and manufactured for agiven application. For clinical samples, a variety of commercial swabtypes are available including nasal, nasopharyngeal, buccal, oral fluid,stool, tonsil, vaginal, cervical, and wound swabs. The dimensions andmaterials of the sample collection devices vary, and the devices maycontain specialized handles, caps, scores to facilitate and directbreakage, and collection matrices. Blood samples are collected in a widevariety of commercially available tubes of varying volumes, some ofwhich contain additives (including anticoagulants such as heparin,citrate, and EDTA), a vacuum to facilitate sample entry, a stopper tofacilitate needle insertion, and coverings to protect the operator fromexposure to the sample. Tissue and bodily fluids (e.g. sputum, purulentmaterial, aspirates) are also collected in tubes, generally distinctfrom blood tubes. These clinical sample collection devices are generallysent to sophisticated hospital or commercial clinical laboratories fortesting (although certain testing such as the evaluation ofthroat/tonsillar swabs for rapid streptococcal tests can be performed atthe point of care). Environmental samples may be present as filters orfilter cartridges (e.g. from air breathers, aerosols or water filtrationdevices), swabs, powders, or fluids.

A common collection technique for forensic evidence is performed using aswab. Swabs are commercially available from Bode (Lorton, Va.), Puritan(Guilford, Me.), Fitzco (Spring Park, Minn.), Boca (Coral Springs,Fla.), Copan (Murrieta, Calif.) and Starplex (Etobicoke, ON, Canada).Swabbing can also be performed using gauze-like materials, disposablebrushes, or commercially available biological sampling kits. Forensicsamples may contain blood, semen, epithelial cells, urine, saliva,stool, various tissues, and bone. Biological evidence from an individualthat is present in person is often collected using buccal swabs. Awidely used commercial buccal swab is the SecurSwab (The Bode TechnologyGroup, Lorton, Va.). Buccal samples are collected by instructing thesubject or operator to place the swab into the mouth on the inner cheeksurface and to move the swab up and down one or more times.

Another major advantage of the biochips of the invention is the abilityto perform complex processes on multiple samples in parallel. It isimportant to note that the biochip's flexibility allows multiple samplesto be processed using the identical set of manipulations or each sample(or subset of samples) to be processed using a tailored set ofmanipulations. In addition, several independent analyses can beperformed on a given sample. For example, a forensic sample could beanalyzed by isolating DNA and then performing STR analysis, SNPanalysis, and mitochondrial sequencing on the purified material.Similarly, a clinical sample could be analyzed by purifying nucleicacids and proteins and performing PCR, reverse-transcription PCR, DNAsequencing, and immunoassays, allowing (for example) a given sample tobe interrogated for a large number of pathogens and cellular processessimultaneously on a single biochip. We describe herein numeroussolutions to different desired functionalities as well as solutions todifferent integration issues. These are provided for illustration andnot by way of limitation, as one skilled in the art will be able toadapt these components and integration techniques to a variety ofbiochips and instruments for a variety of uses.

A series of software and firmware is required for biochip operation anddata analysis. The instrument hardware is controlled by software andfirmware that dictate component function and perform instrumentself-testing. An automated script controls all interactions of theinstrument with the biochip, including the application of all scriptedprocess steps. Analytical software performs both the processing of rawdata (e.g. color correction of an electropherogram) and analysis if theresults of the assay (e.g. fragment sizing, STR allele calling, DNAsequence analysis). The instrument may contain a graphical userinterface that allows the user to initiate the process and inform theuser of process status. Finally, the system may store relevantanalytical comparators (e.g. STR profiles from individuals of interestor DNA sequence of pathogens), or the system may port out results forexternal database matching and further analyses.

Biochip Fabrication

The invention provides methods for fabricating biochips that are fullyintegrated, require no pre-process sample preparation, are not limitedby the SBS size range and can be significantly larger, are not limitedto microtiter formats, incorporate sample number complexity and processcomplexity.

The biochips can be fabricated from thermoplastic polymers includingpolyethylene, polypropylene, polycarbonate, polystyrene, polymethylmethacrylate (PMMA), polyethylene terephthalate (PET), cyclic olefinpolymer (COP), and cyclic olefin copolymer (COC), and other polymerswhich are suitable to mass-production in accordance with the novelmethods claimed herein. The biochips can be fabricated by CNC-machiningof large plates (including those larger than SBS standard plates), andthe blank plates for CNC-machining may be injected molded themselves.The biochips can also be made by injection molding of large plates(including those larger than SBS standard plates). Optionally, thebiochips can be coated (either prior to, at an intermediate step during,or following assembly) to enhance performance. A wide range of coatingsand treatments can be applied. For example, exposing channels with 0.5%bovine serum albumin reduces binding of proteins present in the sampleto channel walls. Hydrophobic products such as PFC 502A (Cytonix,Beltsville, Md.) can be applied to channels, chambers, and valves tominimize bubble formation. Similarly, the elastomeric or non-elastomericmembranes of pneumatic and mechanical valve structures can be coated orotherwise treated to provide optimal sealing properties.

Yet another aspect of this invention is biochips and methods for makingthem that solve the problems of fabricating large biochips to performintricate series of processing steps in parallel to allow multiplesamples to be processed on the same biochip. Whether the biochip isfabricated by CNC-machining or injection molding, this complexity isachieved by packing the fine features densely together, in certainregions of the biochip, leaving space between them to allow bondingwhile maximizing aspect ratios. The injection molded biochips aredesigned and fabricated such that they are flat, suffer minimal warpage,have defined shrinkage characteristics during fabrication and bonding,and have appropriate alignment tolerances to support the features thatallow process step complexity.

The biochips of the invention include several subassemblies includingfluidic subassemblies, pneumatic subassemblies, valve subassemblies,macrofluidic processing subassemblies, and separation and detectionsubassemblies. Not all subassemblies are required for a given biochip.For example, if macrofluidic sample volumes and on-chip reagents are notrequired, the macrofluidic subassembly is not necessary. Nevertheless,the biochips of the invention will generally have at least oneCNC-machined, embossed, extruded, or injection-molded layer. A singlelayer would contain both fluidic features and the control featuresrequired to transfer fluids from one region of the biochip to another.In many cases, a separate fluidic layer and control layer are preferred,allowing for increased process complexity. The layer or layers may havefeatures on either one or both sides; the advantages of dual-sidedfeatures is increased process complexity, feature density, and samplenumber, decreased fabrication cost, and ease of assembly. When multiplelayers are utilized, they may be bonded together using a number oftechniques known in the art such as thermal bonding, solvent bonding,adhesive bonding, and ultrasonic welding (reviewed in Tsao. C.- W.(2009). Bonding of thermoplastic polymer microfluidics. Microfluidicsand Nanofluidics 6: 1-16). In considering the number of layers in amicrofluidic biochip, it is advisable to utilize the fewest number oflayers that allows both the desired complexity to be achieved.Minimizing layer number reduces the complexity of fabrication andassembly and ultimately, the cost of manufacturing the biochip.

Another aspect of the fabrication of the biochips of the inventionconcerns the layers located on the top and bottom of the varioussubassemblies. The purpose of these intermediate layers, termed “thinfilms”, includes bonding the CNC-machined or injection molded layers toeach other, externally sealing the CNC-machined or injection moldedlayers, providing thermal coupling between the instrument and chamberswithin the layers, providing appropriate optical characteristics toallow efficient excitation and detection by components within theinstrument, providing conduits between layers, supporting membranes,filters, and other elements, and providing material for valving intandem with the injection molded layers. Non-elastomeric thin filmmaterials include polyethylene, polypropylene, polycarbonate,polystyrene, cyclic olefin polymer, cyclic olefin copolymer, acrylic,polyethylene terephthalate, aluminum, and cellulose triacetate.Elastomeric thin film materials include a wide range of rubbers(including silicone) and thermoplastic elastomers as described inPlastics: Materials and Processing Third Edition (ibid). These films canrange in thickness from 1 micron to 500 microns.

As the CNC-machined or injection molded layers and thin films are boundtogether to form the biochip, fluid communication between and acrosslayers is provided by through-holes. Through-holes are features thatpass from top to bottom of a given layer to allow a liquid to move fromone layer to another. Through-holes can take essentially any shape andare frequently cylindrical. The biochips of the invention provide largenumbers of through holes to enable sample number complexity and processcomplexity. For example, the injection molded 6-sample biochips ofExample 6 and their 16-sample counterparts contain the followingthrough-holes:

6-Sample 16-Sample Top Pneumatic Film 117 346 Pneumatic Plate 334 848Rigid Patterned Film 131 346 Fluidics Plate 299 1019 Bottom Fluidic Film14 94 Embossed S&D Film 34 94 S&D Cover Film 0 0

Note that “S&D” refers to “separation and injection.” Taken together,these parts of the 6-sample biochip (exclusive of the macrofluidicprocessing subassembly) contain 929 through-holes and the 16-sampleassembly contains 2747 though-holes. The biochips of the inventioncontain 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500,2000, 2500, or more than 2000 through-holes. When total microfluidicfeatures (including through-holes) are considered, the biochips of theinvention have 1000, 2000, 3000, 4000, 5000, 7500, 10,000 or morefeatures.

With a large number of features per layer, the layers of the biochips ofthe invention must be aligned properly to all layers as well as to thevarious sites of instrument interfacing. A factor in alignment concernsthe shrinkage of parts during bonding, and particularly during thermalbonding; the alignment problem is exacerbated by differential shrinkageof parts. The biochips of the invention are designed and assembled toaccount for differential shrinkage. For ease of assembly, the part withthe greatest number of features is utilized as the shrinkage standard.That is to say, it is designed such that, following bonding, its size isthat required for interfacing with the instrument. The sizes of allother parts are scaled such that, following bonding, they align with thestandard plate.

Alignment at the sites of instrument interfacing is achieved by sizingand placing interface features appropriately. For example, the pneumaticinterfaces of the invention consist of several dozen ports. Instead ofbeing spread throughout the pneumatic layer of the biochip, they areconcentrated centrally. Although the pneumatic layer as a whole shrinks,the much smaller pneumatic interface region shrinks much less. Inaddition, the biochips have alignment features for assembly, whichinclude alignment holes incorporated within the fluidic and pneumaticlayers. To align the fluidic to the pneumatic layer, a dowel pin isinserted into the corresponding alignment holes of the pneumatic layerand fluidic layers. Alignment features to ensure appropriate placementwithin the instrument include guides within the instrument whichregister to the pneumatic layer (and not the fluidic layer), where thepneumatic port features are located. Although parts shrinkage can beestimated, it is prudent to measure shrinkage following the actualassembly process. For example, the pneumatic plate and fluidic plate ofExample 5 shrunk by (−0.063%, 0.116% in the X and Y directions,respectively) and (0.151% and 0.401%) respectively, and the pneumaticplate was increased in size by (0.214%, 0.285%) % to compensate for thisdifferential shrinkage.

Both layer-to-layer alignment and biochip to instrument alignment aremanaged by minimizing the number of parts to be aligned. For example,the CNC-machined or injection molded pneumatic and fluidic plates of thebiochips of the invention generally have features present on both sides.This halves the number of parts required for assembly.

In tandem with alignment features, the features of a given layer thatmust communicate with those of another layer are designed withtolerances. In particular, valve features (as described in Example 2require pneumatic features to align with fluidic chambers and valveseats. In addition, through holes in the pneumatic layer connect theoutput of chambers within the macrofluidic processing subassembly tocorresponding input holes within the fluidic layer. In general when twothrough holes need to be aligned, one of the through holes is made 0.5mm in diameter larger to ensure that alignment of the two through holesis achieved.

The biochips of the invention represent closed systems in that thesamples are inserted and sealed into the biochip and no liquids escapefrom the biochip (i.e., the process reagents that are contained withinthe biochip at the initiation of sample processing are contained withinthe biochip at the conclusion of sample processing (and are removed fromthe instrument when the biochip is removed). Air from the pneumaticdrivelines enters the biochip and air exits the biochip through ventmembranes. The closed system biochips of the invention do not expose theoperator to sample or chemicals, and important safety feature, andsimplify the instrument in that neither unused or spent reagents need bestored within the instrument.

Injection molding of microfluidic features presents many challengesbecause of the large number of fine features which must be present in acomplex fully integrated biochip. These microfluidic features aresometimes present on a given part along with macrofluidic features,further increasing the challenges of injection molding. We presentherein specific solutions to design considerations for specific types ofbiochips, but, one skilled in the art will be able to apply thesespecific solutions to a fabricate a number of types of biochips forspecific functionalities. Among the design considerations in theseexamples are: geometrical aspects such as shrinkage and shape stabilityin precision injection molding, flatness of biochips, warping ofbiochips, minimum feature sizes, feature density, and appropriate aspectratios. The design features of the injection molded parts of thebiochips of the invention will be described in the context of theinjection molding process. Injection molding is a process forfabricating plastic parts in which a mold is clamped under pressure toaccommodate the molten plastic injection and cooling process. Plasticgranules (pelletized resins) are fed into the injection molding machine,followed by the appropriate colorants. The resins fall into an injectionbarrel, where they are heated to a melting point and injected into themold through either a reciprocating screw or ramming device. The moltenplastics are contained within the mold, and hydraulic or mechanicalpressure is applied to make sure all of the cavities within the mold arefilled. The plastics are allowed to cool within the mold, the mold isopened, and the plastic part is ejected with ejecting pins. The entireprocess is cyclical, with cycle times ranging from between ten and 100seconds, depending on the required cooling time.

The mold consists of two primary components, the injection mold and theejector mold. Plastic resin enters the mold through a sprue in theinjection mold; the sprue bushing is sealed tightly against the nozzleof the injection barrel to allow molten plastic to flow from the barrelinto the mold cavity. The sprue bushing directs the molten plastic tothe cavity images through channels (or runners) that are machined intothe faces of the plates. The molten plastic flows enters one or moregates and into the cavity geometry to form the desired part. The mold isusually designed so that the completed part reliably remains on theejector side of the mold when it opens. The part then falls freely whenejected. Molds can be manufactured with a combination of machiningmethods including CNC milling and Electrical Discharge Machining.

The overall approach to the design of the inventive biochips forinjection molding include:

-   -   Reduction in the number of layers. The number of injection        molded layers was reduced to one fluidic layer and one pneumatic        layer by fabricating features on both sides of the layers. This        was accomplished in part by routing the fluidic and pneumatic        lines in a manner such that channels that cross over each other        were routed onto separate sides of the layers. On some        occasions, up-down transitions were required. These were        minimized as vertical transitions lead to large pressure drops        and reduction in the channel conductance. Features to allow        ultrasonic welding of vent and filter membranes have been        designed. The incorporation of welded features allowed an        injection molded layer to be eliminated.    -   Increase in feature density. Increasing the packing density of        channels and features reduces the number and size of the        injection molded layers, reducing shrinkage and improving        alignability. In the pneumatic manifold region FIG. 44, 61 of        the pneumatic layer of the biochips of Example 5, for example,        the pitch of channels is 1.0 mm, and 10 channels are located        within a 1 cm² region. This density was accomplished by        developing a thermal bonding procedure that sealed the channels        despite the narrow (0.5 mm) walls between channels. Thermal        bonding of features with fine pitch and narrow walls is        accomplished by using bonding fixtures with uniform surfaces and        low surface roughness to ensure flatness. Furthermore, two        bonding platens were used and set to have be parallel with        tolerance of 0.0005″ to ensure consistent bonding of fine        features across a given injection molded assembly or        subassembly. Finally, temperature uniformity across the platens        to within 0.25° C. also ensures consistent bonding across the        assembly or subassembly.    -   Minimization of draft angles. Vertical sides of all features        have been angled slightly with draft to ease release of the part        from the mold. Draft angles in injection molding typically range        from range from 2-7°. The injection molded plates of the        invention used even smaller draft angles of 0.5%; the smaller        the draft angle, the more densely packed the injection molded        features. To accommodate for these small draft angles, ejector        pins were placed much closer to the microfluidic features than        typical in a molding process.    -   Through holes. The number of knit/weld lines was reduced by        appropriate placement of gates in the injection mold. In        addition, large open features on the molded parts were minimized        as it is difficult to hold feature dimensions and resin flow        around large features.    -   Wall thickness. A 1.0 mm wall thickness at feature floors and        0.5 mm wall thickness between features was maintained throughout        the design.    -   Flatness and Warpage. Appropriate layer thicknesses and        transitions between features to minimize thickness have been        adopted. The flatness of the resulting plate is characterized        and adjustments to the injection molding process to minimize        part internal stresses is performed. Areas of the molded plate        that are warped are adjusted by modifying placement of ejector        pins. In addition, fine machining of the uneven areas improves        flatness.    -   Feature aspect ratio. High aspect ratio features have been        minimized and aspect ratios have been kept below 2. Higher        aspect ratio features tend to prevent the molded parts from        ejecting effectively from the mold tool.    -   Ejection features. Ejector pin locations have been minimized and        placed only on the ejector half of the mold. The ejector pins        generate local deformation in the surface of the part which in        turn can lead to regions that bond poorly or not at all. The        travel of each ejector pin within the mold was adjusted to        minimize the travel required for repeatable part ejection.    -   Injection molding shrinkage. The shrinkage on injection molding        for the resin was thoroughly characterized to accommodate        shrinkage. Furthermore, shrinkage was further fine tuned by        adjusting the molding temperature and hold times. Shrinkage of        the pneumatic and fluidic parts are adjusted such that all        features within the injection molded plates are aligned within        defined tolerances.    -   Block molding. The approaches described above are applicable to        the molding of both flat plates (e.g. the fluidic plate,        pneumatic plate, and macrofluidic processing cover plate). To        mold higher volume blocks such as the macrofluidic processing        block, these approaches are modified in that the aspect ratios        are typically greater than 2. For block molding, draft angles of        less than 1° C. are incorporated into the design and allow        effective pin removal from the molded part while maximizing        volume of the large chambers.

The fully integrated biochips of this invention realize the potential ofmicrofluidics, by providing biochips that can carry out complex seriesof process steps from the insertion of a sample to the generation of aresult, performed in a single instrument without operator intervention.Moreover, these inventive biochips may be disposable and manufacturedcost-effectively.

EXAMPLES Example 1 Reagent Storage and Release

Biochips of the invention require reagents to perform various processes,and these reagents must be compatible with the biochip materials andoperating and environmental conditions to which they are exposed.

The reagents of the invention can be introduced into the biochip inseveral ways. First, reagents can be added to the biochip manually,generally shortly before use. The advantage of manual addition is thatthe biochip need not hold the reagents for extended periods, minimizingthe need for long-term stability on the biochip. The disadvantages ofmanual loading are that the operator must have technical training andexpertise, the reagents can be placed incorrectly preventing properprocessing, and reagent placement can be a source of contamination. Asecond method of placement is by storing the reagents in containerswithin or near the instrument; tubing or other conduits would thentransfer the reagents to the biochip at the desired process time. Anadvantage of this approach is that placing reagent containers once mayallow multiple analytic runs of the system. Disadvantages are the needfor substantial transfer conduits within the instrument, compromisedflow through the conduits as, for example, as residual reagents dry onthe conduit, the need to open the instrument, allowing for the potentialof damage or contamination, a relative increase in the size of theinstrument to allow reagent storage for multiple analytic runs, and theneed for an operator with technical training and expertise.

A preferred approach is to store the reagents within the biochip, withthe pre-loaded reagents being an integral part of the biochip andessentially inaccessible to the operator. The advantage of this approachis that the operator need never come in contact with the reagents orrequire technical training or expertise, and, if the biochip is a closedsystem, the potential for contamination is minimized. Furthermore, theinstrument accepts the biochip but does not have to be opened to placeseparate reagent tubes or cartridges. When combined with a drive systemthat ensures that the on-chip reagents do not come in contact with theinstrument, the biochip is a closed system, further enhancing ease ofuse while minimizing the possibility of contamination.

In order to store reagents on-chip, they must be compatible with allbiochip materials with which they come in contact and must be accessibleto the process when required. We term one approach to these requirements“Reagent Storage and Release (RSR),” indicating that the reagents arestored and sequestered within the biochip and released on demand asrequired during processing.

Approaches to RSR include blister packages, tube-in-tube structures (inwhich reagent-containing tubes are inserted into biochip reagentreservoirs), and single tube structures (in which reagents are filleddirectly into the biochip reagent reservoirs). Actuation methods includepressure-based methods (which may optionally involve the use of scoredfoils), pin-based methods (in which mechanically or pneumaticallyactuated pins puncture foil seals), and mechanical methods (in which aninstrument component exerts pressure on the stored reagent).

Blister Packages.

In this approach, aluminum foil sealed blisters or stick packages areused to store reagents. Mechanical force is applied to the packages torelease the contents into the reagent reservoir.

Tube-in-Tube Structures.

In this approach, reagent tubes are fabricated in a thermoplastic, and afoil seal is applied to the bottom of the tube prior to reagent fillingand a second foil seal applied to the top of the tube following filling.The sealed reagent tube is then inserted into the appropriate reagentchamber of the biochip. A wide selection of laminated aluminum sealingfoils is available for heat sealing. These foils feature a polymericheat sealing surface laminated to a thin aluminum film. Coatings used onaluminum foil for protection are selected for resistance to the reagentsthemselves, heat, and scuffing. The protective coating may also serve asa heat-seal surface.

Single Tube Structures.

In this approach, chambers within the biochip itself are the storagevehicles. In this approach, the advantage is that the biochip isrelatively smaller (as the tube-in-tube requires an additional wall) andis easier to fabricate. FIG. 1 illustrates the single tube RSR approach.Foil seals (101) are bonded (thermally, by ultrasonic welding, or byadhesives) to the top and bottom of each reagent storage chamber (102).The bottom foil is bonded first, liquid reagents are filled (indicatedby shading of 102), and the top foil is then bonded, sealing the reagentstorage chamber. A top cover (103) contains pneumatic drive lines thatprovide pressure required to burst the foils. Lyophilized reagents (104)can also be stored between foils.

The pressure required to burst the top and bottom foils causes reagentsto flow rapidly out of the reagent chamber. In order to reduce reagentflow, flow-control orifices are fabricated at the bottoms of thechambers. The diameter of the orifices are sized to allow the desiredflow at the applied foil bursting pressures. Valves and vent membranescan also be utilized to control fluidic flow and queue liquids asdescribed in Example 2. In addition, when the RSR chambers are subjectedto pneumatic drive pressure, the foil must have enough space to expandprior to bursting. To accommodate this space requirement (approximately1 mm for a foil of 25 micron thickness and diameter of 8 mm), an RSRspacer plate was designed to provide an expansion space for bursting.The RSR spacer plate also provides the interface to couple the largerdiameter (8 mm in this instance) outlets of the reagent chamber and thesmaller diameter (0.5 to 1 mm) inlet holes of the biochip, andtransitions the reagent flow between the two interfaces with tapers. Theinlet holes may be through holes in the pneumatic subassembly leading tofeatures in the fluidic subassembly, or may couple directly to thefluidic subassembly or the separation and detection subassembly. FIG. 2is a photograph of the spacer plate 105 used for a 5-sample biochipshowing RSR transitions 106 to couple liquid reagents chambers whichrequire RSR bursting. Conventional transition 107 couples processchambers utilized for mixing and holding (but not reagent storage asthey do not require expansions space) to the inlet holes of the biochip.FIG. 3 is a cross-sectional view of an RSR transition 106 which iscomposed of a RSR chamber interface 108, a conical taper section 109,and a biochip interface 110. Also shown is a conventional transition 107which is a through hole transition between the process chambers andanother biochip subassembly. The spacer plate is attached to the reagentchambers and to the by pressure sensitive adhesive and mechanicalfasteners. Other forms of attachment include the use of gaskets with andwithout adhesives, screws, heat staking, and rivets.

Pneumatic bursting of foils is the preferred method, as it requires onlypneumatics for actuation. The foil seals are burst by pneumatic pressurethat is delivered to the tops of the tubes via pneumatic drive lineswithin the biochip. In this configuration, the pressure applied to thereagent chamber is imposed on the top foil, causing it to rupture. Onrupture, the pressure is applied directly to the reagents within thetube. This in turn causes the bottom foil to rupture and release thesolution for processing. Several factors must be considered in theselection of foils for pneumatic bursting. To be compatible forpneumatic bursting, the bursting pressure must be appropriate for thestructure of the biochip and tolerated by the biochip. For the biochipsof the present invention, assembled by thermal bonding, solvent bonding,and adhesive bonding, the desired bursting pressure is approximately20-500 psig. Thin foil films of 10-500 microns are appropriate for thispurpose, preferably 10-30 microns.

FIG. 4 shows pneumatic bursting pressures required for 12 (solid line)and 18 (dashed line) micron aluminum foils bonded to chamber diametersof 2-8 mm. Pressure required for bursting increases with increasing foilthickness and decreases with increasing chamber/foil diameter. Eachpoint shows the average pressure required for bursting (For 2 mmdiameter, n=2, for 3 mm diameter, n=6, for 4 mm diameter, n=10, for 5 mmdiameter, n=9, for 6 mm diameter, n=10, for 7 mm diameter, n=4, and for8 mm diameter, n=4). Bursting pressure can also be affected by the typeof foil material.

Optionally, scoring of the thin foils allows a reduction in burstingpressure; scores of various sizes and shapes on the foil can be utilizedas well. For example, laser or mechanical die scoring of lines, crosses,and circles to varying depths can be used to further modulate burstpressure. FIG. 5 shows an RSR unit with six chambers (each with 8 mmdiameter and 50 mm height). A 25 micron aluminum foil was subjected tolaser scoring (crossed 4.5 mm centrally placed lines of depth ofapproximately 10 microns) and thermally bonded to the unit using a heatstaker. The bottom foil was placed initially, the liquid reagents withfood coloring were filled into each chamber, and the top foil was bonded(FIG. 6).

Two-sided bursting of reagent chambers was performed by filling andsealing 2 ml of reagents into the chambers of FIG. 6. The foils used forsealing the chambers were scored (crossed 4.5 mm centrally placed linesof depth of approximately 10 microns) to achieve a nominal burstpressure of 32 psig. Each chamber was attached to an RSR spacer platewhich was in turn attached to a microfluidic channel. The output of themicrofluidic channel was sealed with a vent membrane to allow air fromthe bursting to vent but not to block the fluid that is released fromthe chambers. A cover was fastened to the top of the chamber. The covercouples each of the 6 chambers to a pneumatic drive line. The pneumaticsystem was configured to apply pressure to only one chamber at a time.The pneumatic drive pressure was increased gradually until liquid wasobserved to flow into the microfluidic channel, and this pressure wasrecorded. All of the 24 foils used in the testing (4 RSR units) burstwith a pressure of 32 psig±3 psig. FIG. 7 shows foils after bursting.Foil materials, scoring depth and configuration, and burst pressures arechosen in tandem to avoid having pieces of the foil become separatedduring bursting, potentially blocking exit channels for the reagents.The scored foil presented here does not generate separated pieces offoil.

In a second experiment, the reagent chambers were filled and sealed withscored foils as above. The chambers were attached to an RSR spacer platewhich was attached to a microfluidic channel. The output of each of themicrofluidic channel was sealed with a vent membrane. A cover wasfastened to the top of the chamber and 45 psig of pressure was appliedto all the reagent chambers simultaneously for 1 second. All of thefoils ruptured and reagent was observed to flow into the microfluidicchannels and stop at the vent membrane. The liquid within the channelwas composed of a single plug without bubbles.

Together these experiments demonstrate the RSR including reagent sealingwithin the chambers, pneumatic bursting of sealing foils, control ofbursting pressure with chamber diameter and scoring, reagent transitioninto a microfluidic channel, and queuing of the reagent with a ventmembrane.

A related approach to RSR utilizes mechanical rather than pneumaticpressure to burst the foils. In this method, sharp pins are fabricatedin plastic at the top and bottom of each foil-sealed reagent tube (metalpins can be inserted to serve the same purpose). Both foils are piercedby pins, releasing the reagents at the required process time.

Example 2 Flow Control in Biochips

The biochips of the invention comprise thermoplastic layers that containfine features, and control of fluid flow is required to execute the setof processing steps required to generate the analytic results. Fluidicfunctions that require the use of flow control elements include:

-   -   Directing fluid flows from one feature to another via channels        in the biochip. The scripted use of drive lines, valves, and        vent membranes directs liquid and air flow through specific        channels and features in the biochip. For example, the        purification filter (FIG. 39, 12) is connected by fluidic        channels to reagent and process chambers. The sequence of fluid        flows and valves that control flow into and out of the        purification filter is described by the DNA binding step, Wash,        Dry, Elution steps of Example 5. In these steps, several vent        membranes, valves (FIGS. 39, 46, 47, 43, 42, and 44) and        pneumatic drive lines are used to direct fluid flows through        this chamber.    -   Sealing of chambers. Certain reactions such as thermal cycling        and cycle sequencing require elevated temperatures. A high        integrity seal is important in such settings because the        pressure of the fluid within the chamber increases as the        reaction is heated. A poor seal will result in fluid leaks out        of the chamber and will allow gases within the reaction to        outgas and generate bubbles. Sealing the chambers minimizes        these problems and improves reaction efficiency. For example,        the thermal cycling chambers of Examples 5 and 6 are sealed        during the thermal cycling process by valves V13 and V14 (FIGS.        40, 52 and 53).    -   Reciprocal mixing. Reciprocal mixing is an approach to mixing        based on forcing two liquids against an air ballast and        releasing pressure to allow reverse flow. Repeated application        and reduction of applied pressure accomplishes mixing by moving        solutions to be mixed “back and forth.” This approach is        illustrated in the biochip of Example 6, which uses an air        chamber (FIG. 70, 610) as a spring which is initially compressed        by the fluid by pressure when the fluid is pushed against it,        and then to push the fluid back when pressure is released. The        air chamber is sealed during reciprocal mixing by valve V13        (FIG. 69, 52) where a linearly increasing pressure of 0 to 15        psig and then 15 to 0 psig is applied. After the mixing, V13 is        opened to allow the mixed solutions to flow through into the PCR        chambers.    -   Queuing vent membranes—Variations in flow velocity of multiple        samples being processed in parallel in a given biochip can        result from factors including differences in sample viscosity        (e.g. DNA content) and variations in channel dimension (and        hence conductance). The use of queuing functions accommodates        variations in the fluid flows for multiple samples through        parallel channels within the biochip. In this approach, the        maximum possible time required for a given step is used as the        step time. Samples that arrive at the vent membrane early will        halt and wait while the other samples with slower flow        velocities arrive at the queuing point. This approach provides        tolerance to process multiple samples simultaneously by ensuring        that they are synchronized. Queuing of fluids is practiced in        Example 5 and 6 “Transfer into thermal cycling chamber” by using        a vent membrane (FIG. 40 and FIG. 69, 100) and a valve (V29)        (FIG. 40 and FIG. 69, 99) to halt the PCR solution for multiple        samples at a common position. An alternative but more        complicated approach to synchronizing multiple samples is to        control each sample separately and to incorporate detection        features (e.g. optical, thermal, chemical, mechanical) that        allow the script to receive feedback when a given sample arrives        at a given point on the biochip.    -   Metering chambers—Solutions are metered in multiple instances        through the process flow. An example of metering in Examples 5        and example 6 is the “Eluate metering” step employs a metering        chamber (FIGS. 40 and 70, 8), valve V12 (FIGS. 40 and 70, 51)        and vent membrane VM (FIGS. 40 and 70, 100).    -   Joining of solutions with air plugs in between. During sample        processing, there are instances in which two discrete fluid        plugs from different inputs and separated by air need to be        combined. An example of this is the “joining of the PCR product        and formamide” step. In this step, a joining chamber (FIGS. 41        and 71, 78), Vent membrane, and valves (FIG. 41, Elements 100,        56, 81) are used. The joining function uses the above elements        to receive each of the fluid plugs into the chamber, remove air        between the fluids, and allow the joined fluid plug to advance.        In this step, valves are used to halt liquid and air flow. The        vent membrane is used to vent air between the fluid plugs.    -   Filling of waste chambers. Waste chambers within the biochip are        designed to accept excess solutions, particularly during        metering steps. An example of waste handling is the “meter        formamide” step. In this step, a waste chamber (FIGS. 41 and 71,        77) consists of a chamber that has a vent membrane to seal the        entire top surface and Valve V15 (FIGS. 41 and 71, 54). The        valve and waste design accommodates excess fluids that may be        variable in volume and may contain air bubbles (and therefore        samples that are discontinuous fluid plugs). The use of a fully        vented waste chamber allows air that is present within fluid        plugs to be ejected, and the use of valves allows the waste to        be directed into the chambers and sealed.    -   Passive sample splitting. The use of a passive differential        conductance feature to spit the flows of two fluids that are        separated by air into two paths may be advantageous. In this        design, a single flow path splits in two flow paths that end        with vented chambers. The flow conductance of a first channel is        significantly reduced relative to that of a second channel by        incorporating a passive flow restriction along its fluidic        pathway. The flow restriction generates resistance, and liquids        that flow to the split will flow in the higher conductance        channel until the vented chamber is filled or the vent membrane        is completely wetted. When either occurs, the restriction of        this flow channel is high and additional liquid flows into the        second channel (despite the flow restriction element). This        design is used to passively control flow of fluid between two        paths based on conductance. It is particularly useful when two        liquids in a channel are separated by an air plug; the first        liquid flows toward the first path and the second liquid is        diverted to a second path.    -   Venting of macrofluidic chambers. The “chaotic bubbling” steps        of Example 5 and 6 result in significant agitation of a large        volume of solution. The lysis solution on chaotic bubbling must        be contained within the chamber to prevent the reagent from        leaving the biochip and ending up in the instrument, while large        volumes of air must be exhausted to allow adequate air flows to        facilitate bubbling. This is accomplished by using a vent        membrane in the cover that acts as a barrier to the fluid flow        but allows air from chaotic bubbling to be exhausted. A similar        design is utilized to vent any chamber subjected to chaotic        bubbling or substantial air flow for any reason.    -   Isolation of drive lines—Vent membranes are also incorporated        into the cover and other portions of the biochip to isolate the        drive lines of the instrument from fluids within the biochip.

Valving Structures

The above functions rely on two flow control structures: valves and ventmembranes. The biochip uses valves for flow control to halt or allowflow of fluids within channels. Valves can be passive or active, andpassive valves include in-line polymerized gel, passive plug, andhydrophobic valves. Active valve structures include mechanical(thermopneumatic and shape memory alloy), non-mechanical (hydrogel,sol-gel, paraffin, and ice), and external (modular built-in, pneumatic,and non-pneumatic) valves. The pneumatic and mechanical valve structurescan also make use of either elastomeric or non-elastomeric membranes. Ineither case, the membranes can be treated to provide optimal sealingproperties.

Many types of valve structures can be utilized in the biochips of theinvention, and several types may be incorporated in an individualbiochip. The selection of a valve structure is based primarily on easeof fabrication and assembly in concert with the approach toinstrumentation. For example, mechanical valves controlled by rods thatactuate biochip features would be appropriate in an instrument withsophisticated mechanical alignment and control mechanisms. Similarly,valves based on localized melting of wax require an instrument withcontrolled special heating mechanisms. In Examples 5 and 6, theinstrument is capable of sophisticated pneumatic control, and theincorporate valves types (described below) are based on pneumaticactuation. Other pneumatic and non-pneumatic (e.g. mechanical, liquid,wax, electrical) actuation mechanisms and corresponding valves can beutilized in the inventive biochips.

The top view of a pneumatically actuated elastomeric valve structure isshown in FIG. 8. The top view of shows pressure sensitive adhesive (PSA)tape and an elastomeric membrane for the normally open valve. Across-sectional view of the pneumatically actuated valve for control offluids and air is shown in FIG. 9. Components of the valve structureinclude a valve membrane 202, valve seats 203, pneumatic chamber 204,fluidic chamber 205, double sided PSA tape 206, fluidic through holes208, fluidic channel 209, and pneumatic channel 210. This valve isfabricated by assembling 3 components:

Fluidic Subassembly.

This subassembly contains a channel for fluid 209 (liquid or air) flow.The channel is interrupted by a set of through holes 208 that passthrough to the surface. At the surface these through holes form thevalve seats 203 for the valve assembly. It is fabricated by CNCmachining the channels and through holes in a thermoplastic sheet (FIG.33) and covering both sides with thin plastic films. In this case thethin film plastic is a thermoplastic film that is bonded. The films havethemselves been patterned by CNC machining, and have features includingthrough holes that are aligned to the corresponding features on the CNCmachined layer to provide access to the fluidic sandwich layer.Similarly, the same features within the fluidic and pneumatic layers canalso be fabricated by injection molding.

Pneumatic Subassembly.

This subassembly couples the pneumatic drive of the instrument to thefluidic subassembly to pneumatically drive fluids within the fluidicsubassembly and to pneumatically activate valves within the biochip. Thepneumatic channels 210 couple the pneumatic drive to the valve chamber.This subassembly is fabricated by CNC machining a channel and chambersonto each of the two sides of a thermoplastic sheet (FIG. 44). Thefeatures in the thin plastic films include through holes are aligned tothe corresponding features on the CNC machined layer to provide accessto the pneumatic sandwich layer. Bonding is accomplished thermally butcan also be performed ultrasonically, with solvents, and usingadhesives. Similarly, the same features within the fluidic and pneumaticlayers can also be fabricated by injection molding.

Valve Subassembly

This subassembly is positioned between the pneumatic subassembly and thefluidic subassembly. In this construct, the valve assembly consists ofan elastomeric membrane 202 that is 0.005″ thick. When pneumaticallyactivated, this layer will deflect to control flow of fluids (includingair) within the fluidic layer. A top pressure sensitive adhesive layer206 is used to attach the membrane to the top pneumatic layer. A bottompressure sensitive adhesive layer is used to attach the membrane layer206 to the fluidic layer. The pressure sensitive layers are patterned bylaser cutting. The pressure sensitive adhesive layer can also bepatterned by methods including die cutting or punching. The elastomericmembrane is from 0.005″-0.015″ thick and the material can be rubberssuch as silicone.

The valve was constructed by assembling the three subassembliestogether. The fluidic assembly and pneumatic assembly are fastenedtogether by thermal bonding. Similarly, the pneumatic and fluidic platescan be fastened together by the use of a number of mechanical fastenersincluding screws, rivets, thermal heat staking and ultrasonic welding.

These valves are normally open. Fluids will flow through the channelwithin the fluidic layer, up the through hole, into the valve body, outthe 2^(nd) through hole and back into the channel within the fluidiclayer. When a pressure is applied to the pneumatic channel, the pressuredeflects the valve membrane and pushes this membrane against the valveseats and floor of the valve fluidic chamber. This seals off the pathbetween the through holes and stops flow through the valve. The valvestructure incorporates membrane and PSA elements that have high degreesof compliance such that, on assembly, their compression accommodates forany local variations in flatness of two parts being bonded. Thisaccommodation enhances the ability to form effective seals around thevalves.

An experiment was conducted to assess the valve sealing pressure. Thepneumatic channels of the valve were connected to a pneumatic drive, VD.A volume of dyed liquid (water) was loaded into a portion of the fluidicchannel that coupled to the valve. The input to the fluidic channel wasconnected to another pneumatic drive FD. The pressure of VD was set andheld constant through the experiment. The pressure of FD was graduallyincreased until the fluid flow within the channel was observed. Thepressure at which the fluid began to flow is the burst pressure of thevalve for a given valve sealing pressure. The burst pressure for thevalve for valve sealing pressures of 5, 15, and 22 psig were 3, 13.5 and20 psig respectively. This data shows that a pneumatic sealing pressureof larger than 2 psig that of the fluidic drive pressure is acceptablefor sealing. In the biochips of Examples 5 and 6, the valve sealingpressure is set to a common pressure (22 psig) for most of the processsteps. This is effective in controlling multiple valves simultaneouslyand for controlling flow channels of up to 18 psig of pressure. Althougha fixed valve drive pressure is adequate for most functions, a variablesystem allows the valve pressure to be increased (for cases in which thefluidic drive pressure requirements are high) and to be reduced (priorto opening the valves) to minimize any agitation of the fluids when thevalve membrane is activated.

When the valve is used during vacuum process steps and a fluid is drawfluid through the valve, a vacuum also needs to be applied to both thefluidic and pneumatic line to maintain the valve in an open position. Toclose this valve, pressure is applied as above.

Although all of the valves in the biochips of Examples 4, 5, and 6 arein the normally open configuration, those valves and the several typesof valves in this example can be designed in a normally closedconfiguration. For example, FIG. 10 is a top view of the PSA tape andelastomeric membrane valve for normally closed operation. FIG. 11 is thecross-sectional view of the PSA tape and elastomeric membrane valve fora normally closed configuration. The construction of this valve isdifferent than that of FIGS. 8 and 9 in that the valve seats are raisedsuch that they are in intimate contact with the valve membrane layer inthe unactivated state. The valve membrane with the valve seats onfabrication are placed under tension during fabrication and assembly. Toopen the valve, a vacuum is applied to pull the valve membrane away fromthe valve seats. Note that all valves in this Example (except for thestructure of FIGS. 10 and 11) are normally open. For some applications,it may be advantageous to incorporate one time use valves, namely valvesthat are not designed to be repeatedly opened and closed during theassay process, but rather which, for example, begin the process in anopen state and close once during the assay process.

The top view of a pneumatically actuated valve with a rigid valvemembrane with normally open configuration is shown in FIG. 12. Across-sectional view of the pneumatically actuated valve with a rigidvalve membrane for control of fluids and air is shown in FIG. 13.Components of the valve structure include a valve membrane 202, valveseats 203, pneumatic chamber 204, fluidic chamber 205, fluidic throughholes 208, fluidic channel 209, and pneumatic channel 210. This valve isfabricated by assembling 3 components:

Fluidic Subassembly.

This subassembly contains a channel for fluid (liquid or air) flow. Thechannel is interrupted by a set of through holes 208 that pass throughto the surface. These through holes form the valve seats 203 for thevalve assembly. Alternatively, the channel can be routed along the topside of the fluidic plate and be interrupted as it passes though thevalve. The channel ends form the valve seats for this assembly. Thisassembly is fabricated by injection molding the channels and throughholes in a thermoplastic. In this case, the valve membrane 202 is a thinfilm thermoplastic film that is bonded. The fluidic chamber 205 of thisstructure is fashioned to the shape of the rigid membrane under fulldeflection to effect sealing.

Pneumatic Subassembly.

This subassembly couples the pneumatic drive of the instrument to thefluidic subassembly to pneumatically drive fluids within the fluidicsubassembly and to pneumatically activate valves within the biochip. Thepneumatic channel couple the pneumatic drive to the valve chamber. Thissubassembly is fabricated by injection molding channel and chamberfeatures in thermoplastic (FIGS. 72 and 73). The features in the thinplastic films include through holes are aligned to the correspondingfeatures on the injection molded layer to provide access to thepneumatic subassembly. Bonding is accomplished thermally but can also beperformed ultrasonically, with solvents, and using adhesives.

Valve Subassembly.

This subassembly located between the pneumatic subassembly and thefluidic subassembly. In this construct, the valve assembly consists of athin thermoplastic film. The film can be between 10 to 250 micronsthick. In Example 6, films of thermoplastic of 40 microns, 50 micronsand 100 microns were used. When pneumatically activated, this layerdeflects to control flow of fluids (including air) within the fluidiclayer. The valve was constructed by assembling the three subassembliestogether. The fluidic assembly and pneumatic assembly were bondedtogether thermally or by solvents. Alternatively, a double-sidedadhesive can also be used to adhere the top and bottom assembliestogether. The thin thermoplastic, rigid valve membrane is integral tothe fluidic layer, a significant advantage during biochip assembly.

These valves are normally open. Fluids flow through the channel withinthe fluidic layer, up the through hole, into the valve body, out the2^(nd) through hole and back into the channel within the fluidic layer.When pressure is applied to the pneumatic channel, the pressure deflectsthe rigid valve membrane and pushes this membrane against the valveseats and floor of the valve fluidic chamber to seal off the pathbetween the through holes and stops flow through the valve.

An experiment was conducted to assess the valve sealing pressure byfabricating a valve using the methods above with a 100 micron membrane.The pneumatic channels of the valve were connected to a pneumatic driveVD. A volume of dyed liquid (water) was loaded into a portion of thefluidic channel coupled to the valve. The input to the fluidic channelwas connected to another pneumatic drive FD. The pressure of FD was setand held constant through the experiment. The pressure of VD wasgradually decreased from a high level 80 psig until the fluid within thechannel is observed to flow. The valve sealing pressure required to holdan FD of 1, 2, 3, 4, and 5 psig was 14.6, 28, 54, 65.5, and 81.5 psigrespectively. The rigid membrane used in this valve constructionrequires the application of a higher pressure to seal the fluid flows.Reducing the valve membrane thickness from 100 microns to 50 microns and40 microns substantially reduced the requirements for pressure to sealagainst fluid flows. For vacuum operation in which fluids are drawnthrough the channels, a vacuum is applied to the valve to maintain it inan open position.

FIG. 14 shows a top view of a clamped, normally open elastomericmembrane valve. The cross-sectional view of this pneumatically actuatedvalve for control of fluids and air is shown in FIG. 15. Components ofthe valve structure include a valve membrane 202, valve seats 203,pneumatic chamber 204, fluidic chamber 205, fluidic through holes 208,fluidic channel 209, pneumatic channel 210, and compression rings 212.This valve is fabricated by assembling 3 subassemblies. An advantage ofthis structure is that it is quite simple and does not require PSA tape.

Fluidic Subassembly

This subassembly contains a channel for fluid 209 (liquid or air) flow.The channel is interrupted by a set of through holes 208 that passthrough to the surface. At the surface these through holes form thevalve seats 203 for the valve assembly. It is fabricated by CNCmachining the channels and through holes in a thermoplastic sheet (FIG.33) and covering both sides with thin plastic films (FIG. 36 and FIG.37). In this case the thin film plastic is a thermoplastic film isbonded. The films have themselves been patterned by CNC machining, andhave features including through holes that are aligned to thecorresponding features on the CNC machined layer to provide access tothe fluidic sandwich layer. Similarly, the same features within thefluidic and pneumatic layers can also be fabricated by injectionmolding. A set of valve membrane compression rings are formed around thefluidic chamber 212.

Pneumatic Subassembly

This subassembly couples the pneumatic drive of the instrument to thefluidic subassembly to pneumatically drive fluids within the fluidicsubassembly and to pneumatically activate valves within the biochip. Thepneumatic channels 210 couple the pneumatic drive to the valve chamber.This subassembly is fabricated by CNC machining a channel and chambersonto each of the two sides of a thermoplastic sheet (FIG. 12). Thefeatures in the thin plastic films include through holes are aligned tothe corresponding features on the CNC machined layer to provide accessto the pneumatic sandwich layer. Bonding is accomplished thermally butcan also be performed ultrasonically, with solvents, and usingadhesives. Similarly, the same features within the fluidic and pneumaticlayers can also be fabricated by injection molding. A set of valvemembrane compression rings are formed around the pneumatic chamber 211.The compression rings of the fluidic and pneumatic chambers arecoincident with each other and will align with each other.

Valve Subassembly

This subassembly located between the pneumatic subassembly and thefluidic subassembly. In this construct, the valve assembly consists of asilicone membrane 202 that is 0.005″ thick. When pneumaticallyactivated, this layer will deflect to control flow of fluids (includingair) within the fluidic layer.

The valve is constructed by assembling the three subassemblies together.The fluidic assembly and pneumatic assembly are fastened together bythermal bonding. Similarly, the pneumatic and fluidic plates can befastened together by the use of a number of mechanical fastenersincluding screws, rivets, thermal heat staking, and ultrasonic welding.The pneumatic and fluidic layers are bonded such that the membrane layerbetween the compression rings (212) is compressed. The degree ofcompression will range from 5% to 60% and is a function of the durometerand thickness of the valve membrane. The degree of compression issufficient when the pneumatic drive is sealed within the pneumaticchamber and the fluids do not leak from the fluidic chambers.

Venting Structures

The microfluidic component of the apparatus uses venting membranes tovent air and to serve as a barrier to fluid flows. The venting membranesthat were used in the biochips of Example 5 and 6 were selected based onthe surface tension of the liquid being transported and the surface freeenergy. When a significant differential exists between the two, thecontact angle between the solid surface and the liquid is high andliquid droplets will be repelled from the membrane surface and not foulthe membrane. Another consideration is the air-flow rates through themembrane. It must be sufficiently high to allow unrestricted venting ofair. Typically, for a given material, the differential between surfacefree energy and surface tension increases with decreasing pore sizehowever, the air flow rate is inversely proportional to the port size.For example, the surface tension of water is 73 dynes/cm, isopropylAlcohol 22 dynes/cm, and for oil 30 dynes/cm.

Venting membranes used in the biochip include those made of:Polytetrafluoroethylene (PTFE), a widely used material in medicalventing and gas filtration. It is an inert material that offersexcellent flow properties and high chemical resistance. Dimensionalinstability of cut shapes of this membrane type can cause difficultiesin robotic handling in over-molding operations. PTFE is incompatiblewith gamma or E-beam sterilization because chain scission causes loss ofintegrity when the material is exposed to ionizing radiation;Polyvinylidene fluoride (PVDF) is a durable material that offers goodflow properties and broad chemical resistance. It is available in bothnatural and super-hydrophobic forms; Ultra-high molecular weightpolyethylene (UPE) is a more recent entry into the medical venting andgas filtration market. It is a naturally hydrophobic material thatoffers excellent flow properties and broad chemical resistance; Modifiedacrylic membrane treated to be hydrophobic is an economical choice forventing applications. It is oleophobic, hydrophobic, and chemicallycompatible.

FIG. 16 shows a cross sectional view of a vent membrane configurationused in the biochips of Example 5. The vent membrane (FIG. 16, 213) isincorporated into the structure placing the membrane between thepneumatic and fluidic layers and by thermal bonding. FIG. 17 shows across sectional view of a vent membrane configuration (FIG. 17, 213)that was used in the biochips of Example 6. In this vent membraneconfiguration, the vent membrane is welded to the fluidic layer with aweld ridge.

Example 3 Biochip Interface

The biochips of the invention interface with an instrument. Theinstrument provides all the subsystems required for the completion ofsample analyses including high and low voltage power subsystems, thermalcycling subsystems, pneumatic subsystems, magnetic subsystems,mechanical subsystems, optical subsystems, ruggedization subsystems,process control subsystems, and computer subsystems as shown in FIG. 58.The instrument to biochip interface will involve one or more of thesesubsystems, depending on the microfluidic drive and the series ofprocesses to be performed within the biochip. In the case of theexamples herein, the interface of the biochips and the instrument arepneumatic, electrical, optical, and mechanical as follows:

Pneumatic Subsystem Interface.

The pneumatic subsystem is coupled to the biochip through a pneumaticmanifold. FIG. 18 shows the pneumatic manifold area for the biochip ofExample 5 and consists of a series of pneumatic ports that are locatedon the top side of the pneumatic plate. Five types of ports areincorporated with the pneumatic interface:

Low flow drives. Low flow ports are used to supply pneumatic foractivation of valves and drive of fluidics through the biochip.High flow drives. High flow ports are used to supply air required forprocesses such as agitation by bubbling and drying of the purificationmembrane, requiring flows of up to 19 SLPM. The port sizes are enlargedto minimize pressure drops.High pressure drive. This port is used to supply up to 400 psig requiredfor sieving matrix filling. Condensate ports. Condensate generated bymechanical pumps within the instrument can optionally be driven into achamber of the biochip through the pneumatic interface.

Instrument Test Ports.

Test ports can be incorporated into the pneumatic manifold for testingof the instrument pneumatic system and confirming alignment of thepneumatic manifold of the instrument to that of the biochip.

The pneumatic ports are positioned in an array, with 34 low flow portsand 7 high flow ports. The location of this pneumatic manifold wasselected based in part on the following considerations:

Minimizing the Surface Area of the Interface Site.

The more compact the pneumatic interface region, the less that localshrinkage and warping during injection molding will interfere with theinterface, allowing improved alignment.

Collecting all Pneumatic Ports at a Single Interface Site.

This approach was taken to simplify the instrument, an advantage in asystem that is ruggedized and will be utilized outside the laboratory.Typically, the single interface will be centrally located within thebiochip footprint, but it may also be located at an end of the biochip.Alternatively, multiple interfaces could be distributed across thebiochip. In the biochips of Examples 5 and 6, the pneumatic ports arecentralized in one location to allow for a compact coupling pointbetween the instrument and biochip. This organization and positioning ofthe manifold simplifies routing of the pneumatic lines within thebiochip as compared to either having decentralized ports or having allports located at an extreme end of the biochip. This also increases thetolerance of the alignment between the manifold and the pneumatic portsand simplifies routing of the pneumatic tubing within the instrument.

Location of Ports on the Biochip Based on Feature Density

There are areas on the fluidic and pneumatic plates where the featuredensity is low. It is optimal to locate the pneumatic manifold in theseareas when possible. In general, optimal use of space on the biochip(i.e., the rational packing of all microfluidic and macrofluidicfeatures) is important to maximize the feature density and processcomplexity of a biochip of given dimensions. In the biochips of Examples5 and 6, the pneumatic ports are located on the top side of thepneumatic layer. Similarly, the pneumatic ports can also be located onthe bottom side of the fluidic layer and be coupled to the instrumentthrough the chip holder. The absolute location of the ports isdetermined by feature density, functional utility, and ease of design.In the biochips of Examples 5 and 6, the thermal cycling regions arelocated on the bottom side of the fluidic subassembly, requiringclamping pressure from above to establish efficient thermal contact withthe thermal cycler. Accordingly, the centralized pneumatic ports werepositioned on the top side of the pneumatic subassembly such that asingle clamping mechanism could function to simultaneously interfacewith the biochip thermally and pneumatically.

Closed System

As noted above, the biochips of the invention are closed systems suchthat all liquids are located within the biochip during and afterprocessing. Vent membranes (as described in Example 2) are used to blockfluid from inadvertently flowing from the biochip into the pneumaticmanifold of the instrument.

As an illustration of pneumatic flow and the pneumatic manifold, theapproach to driving fluids within the macrofluidic processing subsystemfor the “lysis step” of Example 5 will be presented. To effect thisstep, the lysis drive line DL1 (FIG. 39, 68) was activated to drive thelysis solution from the lysis reagent chamber (FIG. 39, 20) into theswab chamber (FIG. 39, 19). The process controller activated thesolenoid relay on the instrument which applied the desired pressure onthe drive line DL1 port on the pneumatic interface (FIG. 39, 20). Air isdriven through the pneumatic interface at the DL1 port (FIG. 18, 301) ofthe pneumatic manifold, along pneumatic channel of the pneumatic plate(FIG. 18, 302) to the microfluidic interface port within the pneumaticplate (FIG. 19, 303). This interface port routed the pneumatic drivepressure through the pneumatic channels within the macrofluidicprocessing block (FIG. 47, 26) and into the cover of the macrofluidicprocessing subassembly (FIG. 48, 27). The cover in turn routed thepneumatic drive along a channel (FIGS. 48 and 53, 304) into the lysisreagent chamber (FIG. 47, 20). Taken together, the resultant step is thetransfer of the lysis reagent from the lysis reagent chamber through thefluidic subassembly, and ultimately, to the swab chamber (FIG. 20, 20).

Similarly, if the reagents within the chamber are sealed by foils as inExample 1, then the pneumatic drive is applied in a two step action,where the first step is to generate a pulse to burst the foil, and thesecond step is similar to the non-RSR illustration above.

High Voltage Subsystem Interface

The high voltage subsystem is coupled to the biochip through a set ofelectrode pins and a wiring harness. Alternatively, a set of etched thinmetal electrodes can also be fabricated. In this setting, electrode pinsare inserted into the anode and cathodes by press fitting into thebiochip. The electrode pins make contact to electrode strips located onthe top of the pneumatic subassembly and also inserted by press fitting.This electrode strip is coupled to the instrument by a set of springloaded electrodes on the instrument. The electrode strips are fabricatedof Beryllium Copper (BeCu), a high performance metal which can befabricated into a wide variety of components. Its mechanical andelectrical properties make it the ideal material for EMI/RFI shieldingproducts. The electrode strip can also be fabricated from other metals.

Optical Subsystem Interface

As described in patent application Ser. Nos. 12/396,110, published as2009/00229983 entitled “Ruggedized Apparatus for Analysis of NucleicAcid and Proteins,” and 12/080,745, published as 2009/0020427 entitledPlastic Microfluidic Separation and Detection Platforms,” both of whichare incorporated herein by reference, the laser is coupled to theseparation and detection window of the pneumatic-valve-fluidic stack(Examples 5 and 6) through an opening in the chip holder. A set ofphotodiodes is used to enable lane finding of the lanes within theseparation and detection biochip.

Thermal Subsystem Interface

The thermal cycler and biochip are positioned such that the thermalcycling chambers are centered within the TEC elements, as described inapplication Ser. No. 12/080,746, published as 2009/0023603 entitled“Methods for Rapid Multiplexed Amplification of Target Nucleic Acids,”which is incorporated herein by reference. A clamping force of 90 lbs isrequired to achieve good contact between the thermal cycling chambersand the TEC to accurately generate the temperature cycling profiles. Theclamping pressure is generated by the clamp arm and compression of asilicone pad. The clamping arm exerts the pressure required to push downon the silicone pad.

An experiment to assess the clamping force required for efficientthermal transfer was performed. A pneumatically activated cylinder (FIG.21, 305) was used to exert a controlled force onto a PCR biochip (FIG.21, 401). At each level of force, the PCR was performed followingstandard protocols (See, Giese, H., et al. (2009). “Fast multiplexedpolymerase chain reaction for conventional and microfluidic short tandemrepeat analysis.” J Forensic Sci 54(6): 1287-96, which is incorporatedherein by reference), and STR profiles were analyzed. The data showedthat at a force of below 75 lbs, inconsistent STR profiles weregenerated. When forces above 75 lbs were applied, consistent andeffective amplification was observed (FIG. 22).

The separation and detection subsystem is coupled to the heaters on thechip holder to maintain a temperature of 50° C. Temperature uniformityis important and the clamping of the biochip onto the heater platescontributes to the establishment and maintenance of temperatureuniformity.

Mechanical Alignment

To align all biochip to instrument interfaces, the biochip is placedonto the chip holder of the instrument containing a set of mechanicalalignment guides. These guides provide alignment of the interfacefeatures of the biochip to the instrument to within ±0.020″. The guidesregister the chip at three locations along the edge of the pneumaticplate, near to the corners to locate the origin and orientation. In thisconfiguration the guides have a cutout to locate only against thepneumatic layer (this minimizes any effect of slight misalignmentsbetween the fluidic and pneumatic plate layers).

Example 4 CNC-Machined Biochip that Accepts and Mixes DNA and PCRReagents and Performs 16-Plex Amplification Reaction in 17 Minutes forSixteen Samples Simultaneously

The PCR biochip 401 of FIG. 23 was injection molded in a slide formatand successfully tested for rapid multiplexed PCR. This biochip is 25mm×75 mm×1.1 mm thick. The system allows multiplexed amplification onSTR fragments from a single genome equivalent of human DNA (6 pg of DNA,essentially a single-copy limit of detection) as described in Giese, H.,et al. (2009). “Fast multiplexed polymerase chain reaction forconventional and microfluidic short tandem repeat analysis.” J ForensicSci 54(6): 1287-96.

Based on that result, a biochip (footprint of 76.2 mm×127 mm) wasdesigned and fabricated by CNC-machining Fluidic layer 1 (FIG. 24, 403)and fluidic layer 2 (FIG. 25, 404), pneumatic layer 1 (FIG. 26, 405) andpneumatic layer 2 (FIG. 27, 406) are fabricated by CNC machining from athermoplastic sheet. The fluidic subassembly (FIG. 28, 407) wasfabricated by assembling fluidic layer 1 (FIG. 24, 403), fluidic layer 2(FIG. 25, 404) and an unpatterned thin thermoplastic film and thermallybonding. The pneumatics subassembly (FIG. 29, 408) was fabricated bythermal bonding pneumatic layer 1 (FIG. 26, 405), pneumatics layer 2(FIG. 27, 406). The biochip assembly (FIG. 30, 409) was fabricated bybonding the fluidics subassembly (FIG. 28, 407) to the pneumaticsubassembly (FIG. 30, 409) and incorporating an elastomeric, normallyopen valve subassembly as described in Example 2.

To test the biochip, 160 microliters of PCR reagent master mix,sufficient for 16 samples of amplification, was prepared and insertedinto the master mix reagent reservoir (FIG. 28, 410) of the CNC-machinedbiochip. 9.3 microliters of DNA (total template of 1 ng) was insertedinto each of the sample wells (FIG. 28, 411). The biochip was connectedto the pneumatic/thermal cycling instrument (FIG. 31). An automatedscript with the following steps was executed:

-   -   Initialization. All valves were closed.    -   Queue sample and reagent. PCR reagent was pneumatically driven        into the PCR metering chambers of the biochip (FIG. 28, 412) by        applying 1 psig to drive line DR (FIG. 28, 419). A volume of 9        microliters of master mix was metered. The sample for each lane        was queued at a vent membrane by applying 1 psig to drive line        DS (FIG. 28, 418).    -   Remove excess PCR reagent. Excess PCR master mix was removed        from the distribution channels by flowing into the reagent waste        chamber (FIG. 28, 414) by opening valve WV (FIG. 28, 413) and        applying 1 psig to drive line DR (FIG. 28, 419). The reagent        waste chamber (FIG. 28, 414) was sealed by closing valve WV        (FIG. 28, 413) and drive line DR (FIG. 28, 419) was deactivated.    -   Move reagent into joining chamber (JC). The PCR master mix was        pneumatically driven into the joining chamber (FIG. 28, 415) by        opening valves RV (FIG. 28, 416) and applying a pneumatic drive        pressure of 1 psig to DR (FIG. 28, 418) for 30 seconds. Reagent        valves RV (FIG. 28, 416) was closed and DR (FIG. 28, 418) was        deactivated.    -   Move sample into joining chamber (JC). Sample was pneumatically        driven into the joining chamber (FIG. 28, 415) by opening valves        SV (FIG. 28, 415) and applying a pneumatic drive pressure of 1        psig for 30 seconds to DS (FIG. 28, 419). The sample and PCR        master mix were joined together in the joining chamber to form        the PCR solution. Valve SV (FIG. 4.6, 415) is closed and drive        line DS (FIG. 4.6, 419) was deactivated. 4.6, 419).    -   Move PCR solution into mixing chamber (MC). The PCR solution was        into the mixing chamber (FIG. 28, 421) by opening Valve JCV        (FIG. 28, 420) and RV (FIG. 28, 416) and applying a pressure of        1 psig for 30 seconds to DR (FIG. 28, 418).    -   Mix the sample and reagent. Reciprocal mixing was performed by        driving the PCR solution into an air chamber AC (FIG. 28, 422)        by opening valves RV (FIG. 28, 416; valve JCV is already open)        and applying a drive pressure to DR (FIG. 28 418) that linearly        increases from 0-15 psig and then from 15-0 psig (over 30        seconds). This was repeated 2 times. Valves JCV (FIG. 28, 420)        and RV (FIG. 28, 416) were closed and DR (FIG. 28, 418) was        deactivated.    -   Move PCR solution into PCR chambers (PC). The PCR solution was        moved into the PCR chamber (FIG. 28, 423) by opening valves PV1        (FIG. 28, 424), PV2 (FIG. 28, 425), JCV (FIG. 28, 420) and RV        (FIG. 28, 416) and applying a pressure of 0.5 psig for 30        seconds to drive line DR (FIG. 28, 418). Valves PV1 (FIG. 28,        424), PV2 (FIG. 28, 425), JCV (FIG. 28, 420) and RV (FIG. 28,        416) were closed.    -   Thermal cycle. Valves PV1 (FIG. 28, 424) and PV2 (FIG. 28, 425)        were closed, and thermal cycling was performed with a 28 cycle        protocol that required 17 minutes.    -   Retrieve PCR product. Valves OV1 (FIG. 4.6, 426), PV1 (FIG. 28,        424), PV2 (FIG. 28, 425), JCV (FIG. 28, 420) and RV (FIG. 28,        416) were opened. A pipette tip was used to manually retrieve        PCR product from port OP (FIG. 29, 427). PCR products were        retrieved from the 16 biochip samples.

Results.

Following the execution of the script, the PCR reaction was removed fromeach channel and separated and detected on Genebench. A representativeSTR profile for the reaction is shown in FIG. 32.

Example 5 CNC Machining of a Fully Integrated Biochip that PurifiesNucleic Acids, Amplifies the Purified DNA, Electrophoretically Separatesthe Amplified DNA and Generates an STR Profile Using an Automated Script

A stationary, unitary plastic biochip that accepts 5 buccal swabs andgenerates STR profiles consists of the following parts:

Fluidic Subassembly

This subassembly transfers and processes fluids within the biochip,interacting with the pneumatic subassembly, macrofluidic processingsubassembly, valve subassembly, and separation and detectionsubassembly. The fluidic plate of the fluidic subassembly was fabricatedby CNC machining an interconnected set of channels and chambers ontoboth the top and bottom sides of a COP sheet. The fluidic plate hasdimensions of 276 mm×117 mm×2.5 mm. FIG. 33 shows the top side of thefluidic plate 1, FIG. 34 shows the bottom side of the fluidic plate 1,and FIG. 35 shows a transparent view of the fluidic plate 1, showingfeatures from both sides. The CNC-machined fluidic plate was fabricatedby machining an injection-molded blank with a thickness of 2.5 mm. Boththe top and bottom sides of the plate are covered with patterned thinplastic films. FIG. 36 shows the top patterned thin film 2 and FIG. 37shows the bottom patterned thin film 3; the thin films have a thicknessof 100 microns. An advantage of having features present on both the topand bottom of the fluidic plate is that only one fluidic plate isrequired for the assembly (two plates would be required otherwise). Ingeneral, minimizing the number of plates in an assembly is a majoradvantage in that it makes alignment of parts more straightforward andreliable, minimizes the effects of differential warpage and shrinkage,and thereby allows more effective assembly at reduced cost.

The channels in this biochip can be characterized as follows: 1) Sampleprocessing channels. Each buccal swab to be analyzed corresponds to aunique set of channels that perform DNA purification, PCR amplification,separation and detection, and all the process steps required to conductthese nucleic acid manipulations. The unique set of channels for eachsample do not communicate with the sets of channels for other samples.That is to say, channels do not communicate from sample to sample and donot share chambers (other than an anode chamber), filter regions, ormembrane regions related to other samples, thereby preserving the purityof each sample and avoiding inadvertent sample mixing or crosscontamination. Certain pneumatic drive lines split to drive multiplessamples in parallel, but the driving is accomplished pneumatically,preserving the integrity of each sample. Moreover, the unique set ofchannels for each sample is a closed pathway; only air form thepneumatic drivelines enter the biochip, and only air through ventmembranes leave the biochip-liquid reagents are provided by the biochipand liquids do not leave the biochip. FIG. 38 shows the path 4 a singlesample takes through the fluidic plate. FIG. 39 shows an expanded viewof this path 4 through the purification region 5, FIG. 40 shows anexpanded view of this path 4 through the amplification region 6, andFIG. 41 shows an expanded view of this path 4 taken through theseparation and detection region 7. It is important to note that thedirection of electrophoresis is opposite to that of purification andamplification. This direction change is facilitated by through holes inthe fluidic layer that pass sample to the separation and detection layerjust below. 2) Gel processing channels. These channels are utilized tofill the separation and detection subassembly with sieving matrix andbuffer in preparation for separation and detection. That is to say, thechannels are filled from common reagent reservoirs. After purification,amplification, and pre-electrophoresis are complete, each separationchannel receives a unique sample via sample-specific cathodes. Eachsample has a unique cathode chamber and separation channel, and allsamples share a single anode chamber through which electrophoresisreagents are loaded.

The fluidic plate contains a particulate filter region, a purificationmembrane region, and several types of chambers, including meteringchambers, reconstitution chambers, mixing chambers, joining chambers,waste chambers, and cathode and anode chambers. These chambers andregions are in line with the channels and have shapes and dimensionsdetermined by the required functionality. For example, a reagentmetering chamber (FIG. 40, 8) is characterized by different dimensionsand volumes than a cathode chamber (FIG. 41, 9) or a reconstitutionchamber (FIG. 40, 10).

The channels, chambers, and filter and membrane regions are covered, inthis case by bonding thin thermoplastic films (CNC-machined or injectionmolded plates can also form the covers). The sandwich of thinfilm-fluidic layer-thin film is termed the “fluidic subassembly.” Thefilms have themselves been patterned by CNC machining, and the thinfilms of the invention can also be patterned by a number of processesincluding die cutting or punching and laser cutting. These thin filmshave features including through holes that are aligned to thecorresponding features on the CNC machined fluidic layer to provideaccess to the features of the fluidic layer. For example, sample passesfrom the fluidic layer through the through holes in the bottom patternedthin film to the separation and detection subassembly. Prior to bondingthe top and bottom thin films to the plate, additional components arealso incorporated for functions including particulate filtration (FIG.39, 11), DNA purification (FIG. 39, 12), and venting (FIG. 41, 13).Bonding of the patterned thin films to the fluidic plate wasaccomplished thermally but can also be performed ultrasonically, withsolvents, and using adhesives. Features on the plate and films can beadded to facilitate the bonding method; for example, energy directorridges can be placed at the sites of ultrasonic welding.

Pneumatic Subassembly

This subassembly couples the pneumatic drive of the instrument (asdescribed in Example 3), to the fluidic subassembly, macrofluidicprocessing subassembly, valve subassembly, and separation and detectionsubassembly to pneumatically drive fluids and activate valves within thebiochip. This subassembly is fabricated by CNC machining aninterconnected series of pneumatic drive lines onto each of the twosides of a COP sheet. Some of the drivelines are sample-specific andothers are related to non-sample specific functions such as gel filling.FIG. 42 shows the top side of the pneumatic plate 14, FIG. 43 shows thebottom side of the pneumatic plate 14, and FIG. 44 shows a transparentview of the pneumatic plate 14, showing features from both sides. Thepneumatic plate has dimensions of 276.7 mm×117.3 mm×2.50 mm. TheCNC-machined pneumatic plate was fabricated by machining aninjection-molded blank with a thickness of 2.5 mm. The top side of thepneumatic plate is covered with a patterned thin plastic film 15 (FIG.45). The bottom side of the pneumatic plate is covered with an intactthin film 16, and certain regions of this film are cut out followingbonding (FIG. 46) to allow access to the plate from the valvesubassembly. Both the patterned and unpatterned thin films have athickness of 100 microns. Patterns can be cut prior to bonding (as inthe case of the top pneumatic film) or following bonding (as is the casewith the bottom pneumatic film). The reason a post-bonding patterningstep was selected was because the large cut-out regions were readilyremoved post-bonding. Bonding of the thin films to the pneumatic platewas accomplished thermally but can also be performed ultrasonically,with solvents, and using adhesives. Features on the plate and films canbe added to facilitate the bonding method; for example, energy directorridges can be placed at the sites of ultrasonic welding. Finally, thepneumatic plate can also have fluidic features and vice versa. This canbe useful if, for example, the fluidic plate is saturated with featuresand the pneumatic plate has available space. The pneumatic plate in thiscase would still be referred to as a pneumatic plate because of itsposition in the overall biochip and the fact that the majority of itsfeatures were pneumatic. Regardless of their location, pneumatic linesand channels contain only air. In contrast, fluidic features containliquids and air.

The features in the thin plastic films include through holes that arealigned to the corresponding features on the CNC machined pneumaticlayer to provide direct access to the pneumatic layer or indirect accessto the fluidic layer via the pneumatic layer. For example, reagents inchambers of the macrofluidic processing subassembly pass through thethrough holes of the pneumatic subassembly (thin films and pneumaticplate) on their way to the fluidic subassembly. Bonding of the patternedthin films to the pneumatic plate was accomplished thermally but canalso be performed ultrasonically, with solvents, by clamping, and usingadhesives.

Valve Subassembly

This subassembly is located between the pneumatic subassembly and thefluidic subassembly. It consists of an elastomeric material, which, whenpneumatically activated, will deform, thereby stopping flow of fluids(including air) within the fluidic layer. This subassembly consists ofan elastomeric film that can be deflected, but any of the valveassemblies described in Example 2 can be incorporated.

Pneumatic-Valve-Fluidic Stack

This subassembly is fabricated by assembling the pneumatic subassembly,valve subassembly, and fluidic subassembly, and thermally bonding thestack together. Vent membranes (FIG. 44, 17) are incorporated into thissubassembly by placing them over the venting features of the fluidicsubassembly (FIG. 35, 13) and corresponding features in the pneumaticsubassembly. The vent membrane is in the form of a single strip that isused to cover the venting features of multiple samples simultaneously.One vent membrane strip is applied to cover many venting areas. Ingeneral, reduction in part counts allows for ease of assembly, whethermanual or automated. Bonding of the two subassemblies can also beperformed thermally, ultrasonically, with solvents, by clamping, andusing adhesives. Features on the plate and films can be added tofacilitate the bonding method; for example, energy director ridges canbe placed at the sites of ultrasonic welding or an additional bondinglayer can be used to facilitate thermal bonding.

Macrofluidic Processing Subassembly

This subassembly consists of two major elements: a macrofluidic blockand a cover. This subassembly accepts samples, provides reagents to thebiochip, and serves as the interface between microfluidic andmacrofluidic volumes and processes, as further described in applicationSer. No. 12/699,564 entitled “Nucleic Acid Purification,” which isincorporated herein by reference. FIG. 47 shows the macrofluidic block18 with swab 19, lysis solution 20, ethanol 21, wash solution 22, andelution solution 23 reagent chambers, and fluidic processing chambersfor lysate holding 24, eluate homogenization 25, air 58, TTE 59, andformamide 60 chambers. The dimensions of the block are 102.8 mm×50.5mm×74.7 mm. The macrofluidic block utilizes volumes from 0.15-3.0 ml andinterfaces with the pneumatic subassembly and the cover. The side of theblock contains pneumatic drive lines 26; these originate at thepneumatic interface of the pneumatic subassembly and pass through it tothe block. From the block, the drive lines pass to the cover. While someembodiments, including the embodiment described in this Example 5,include the macroprocessing subassembly, not all biochips in accordanceto the technology described herein include the macroprocessingsubassembly. For example, a sample of DNA or purified cells could beinjected into a biochip including the pneumatic subassembly and thefluidics subassembly connected by the valve assembly.

FIGS. 48-54 show the cover. The cover consists of three layers. FIG. 48shows the top of cover layer 1 27, FIG. 49 shows the bottom of coverlayer 1 27, and FIG. 50 shows a transparent view of cover layer 1 27.FIG. 51 shows cover layer 2 28. FIG. 52 shows the top of cover layer 329, FIG. 53 shows the bottom of cover layer 3 29, and FIG. 54 shows atransparent view of cover layer 3 29. The dimensions of the assembledcover are 50.8 mm×118.4×4.5 mm. The cover functions to bring pneumaticdrive lines from the side of the block to the top of the macrofluidicchambers to facilitate reagent transfer, bubbling, and fluidicprocessing. The cover also has venting features associated with the swabchambers 30 and Eluate holding chambers 31. The vent membranes isolatethe chambers in that they do not allow fluid to escape while allowingair to escape to normalize pressure. The cover also serves to hold theswab in place. Optionally, the swab cap or cover can incorporate alocking mechanism to hold the sample in place.

The macrofluidic block and cover components were fabricated by CNCmachining a thermoplastic. Alternatively, these parts can be fabricatedby injection molding and compression injection molding and extrusion. Tomold the macrofluidic processing block aspect ratios are greater than 2and draft angles of less than 1° C. are incorporated into the design.The macrofluidic block was attached to the cover by clamping using agasket and screws. The block is attached to the pneumatic subassemblyusing double-sided PSA. Alternatively, the block attachments can be madeby solvent and thermal bonding.

Additional approaches to macrofluidic processing are described inpending application Ser. No. 12/699,564 entitled “Nucleic AcidPurification,” incorporated by reference herein. The macrofluidicprocessing assembly offers substantially flexibility to the applicationsof the biochip. The liquid reagents and their volumes can be readilymodified based on the types of sample manipulations being performed. Theswab chamber can be modified to accept a wide variety of samplesincluding different types of swabs, blood samples, and environmentalsamples. In certain circumstances, these changes can be made withminimal changes to the pneumatic-valve-fluidic stack; for example,simply changing the lyophilized cakes in the stack would allow anentirely different amplification assay to be performed on a differentsample type.

Separation and Detection Subassembly

This subassembly is used for the separation and detection of nucleicacids and consisted of 16 microchannels, each with an injection andseparation portion. Detailed description of separation and detection inplastic biochips is found in application Ser. No. 12/080,745, publishedas 2009/0020427 entitled “Plastic Microfluidic Separation and DetectionPlatforms,”, incorporated herein by reference. Two approaches to sampleinjection have been utilized, pressure loading and cross injection. FIG.55 shows the separation and detection biochip for pressure injection 32.It functions by pneumatically driving the sample for separation anddetection to the start of the separation column for electrokineticinjection. The cross-sectional dimension of the separation channel (90microns wide and 40 microns deep) and length of the channel between theanode and the cathode (24 cm) was equal for all channels. Thissubassembly was fabricated by embossing onto a thin thermoplastic sheet.Alternately the subassembly can be made by extrusion or injectionmolding. Through holes were fabricated within the embossed sheet toprovide access to the channels from the microfluidic subassembly. Thechannels were covered by bonding a thin thermoplastic sheet over them.

FIG. 56 shows the separation and detection biochip for cross injectionfor electrokinetic transport of the sample for separation and detectionto the start of the separation column for electrokinetic injection 33.The dimensions of the biochip are 77.7 mm×276 mm×376 microns. Thecross-sectional dimension of the separation channel (90 microns wide and40 microns deep) and length of the channel between the anode and thecross-injector (25 cm) was equal for all channels. Six of the 16channels were active in this subassembly. The separation lengths(distance between the intersection and the excitation/detection window)for each of the channels range from 16 to 20 cm long. Thecross-sectional area of the channels between the cathode well and theinjector was adjusted such that all the electrical resistances, andhence electric fields between the cathode and the intersection areessentially equal under bias. This ensured that the electric fieldsexperienced by the samples were identical regardless of the separationchannel into which a sample was loaded. The intersection voltages forall channels were essentially identical. The sample inlet and samplewaste arms for sample injection were both 2.5 mm long. The offsetbetween both channels was 500 microns. This subassembly was fabricatedby embossing onto a thin thermoplastic sheet. Alternatively, thesubassembly may be fabricated by extrusion or injection molding. Throughholes were fabricated within the embossed sheet to provide access to thechannels from the microfluidic subassembly. The channels were covered bysolvent bonding another thin thermoplastic sheet. Alternatively, thesheet can be attached by thermal bonding or thermal-assisted solventbonding.

The separation and detection subassembly (whether pressure loading orcross injection format) was attached to the fluidic subassembly usingadhesive tape, and the subassemblies were oriented such that nucleicacid flow in within the separation and detection biochip was conductedin the opposite direction as NA purification and amplification. That isto say that the nucleic acids traveled through the separation anddetection subassembly in the reverse direction from the general flow offluids during purification and PCR amplification. This reverse ofdirection by using opposite sides of the subassembly allows the complexbiochip to have a substantially reduced length. FIG. 57 illustrates howall the layers—the macrofluidic cover 27, 28, and 29; the macrofluidicblock 18, pneumatic layer with top and bottom films 13, 15 and 16; valvesubassembly layer; fluidic layer with top and bottom film 1, 2, and 3;and separation and detection biochip 32 or 33 are stacked together toform the biochip. FIG. 58 is a photo of the biochip assembly 34 witharrows indicating the direction of process flows.

With reference to FIGS. 39 and 41, to run the biochip the followingreagents were loaded into the macrofluidic processing subassembly priorto the test:

-   -   Lysis solution (FIG. 39, 20), 550 microliters—Chaotropic salt        based reagent for lysing cells and releasing DNA from samples.    -   Ethanol (FIG. 39, 21), 550 microliters—Together with the lysis        solution, lyses cells and facilitates DNA binding to the silica        based purification filter.    -   Wash solution (FIG. 39, 22), 3 ml—Ethanol based reagent for        washing the purification filter to remove proteins and other        biological materials to generate pure DNA.    -   Elution solution (FIG. 39, 23), 300 microliters—Tris-EDTA based        reagent that releases the purified DNA from the purification        filter and stabilizes the DNA.    -   TTE buffer (FIG. 41, 58), 1.6 ml—Tris-Taps-EDTA based reagent        for electrophoresis.    -   Formamide (FIG. 41, 59) 150 microliters—Denaturation solution        for diluting PCR product to prepare the sample for separation        and detection. By mixing sample and formamide at a ratio of        approximately 1:4 (range from 1:50 to 1 to 1.5), it is not        necessary to perform heating or snap cooling to effect        denaturation. The obviation of the conventional heat/cooling        steps simplifies the instrument.

With reference to FIGS. 40 and 41, the following lyophilized reagentswere loaded into the fluidic subassembly of the biochip prior to thetest:

-   -   PCR cake FIG. 40, 10—Lyophilized PCR reaction mix.    -   Internal lane (sizing) standard (ILS) cake FIG. 41,        35—Lyophilized DNA fragments that are fluorescently labeled to        allow the electrophoretic mobility of the amplified fragments to        be correlated to molecular weights.

An advantage of this approach is that all lyophilized reagents arelocated within one subassembly. This allows for improved supply chainmanagement as the conditions for filling lyophilized cakes (low humidityand static) are quite different from and much more demanding than thosefor filling liquids. Accordingly, liquid and lyophilized reagents can befilled separated and in parallel. Optionally, a lyophilized cakecontaining allelic ladder and ILS fragments can be inserted into the topcake chamber 35 for STR analytic runs.

The following reagent was loaded into the separation and detectionsection of the biochip prior to the test:

-   -   Sieving matrix (FIG. 55, 36 and FIG. 56, 36)—High molecular        weight linear polyacrylamide solution for electrophoretic        sieving of DNA.

The biochip was inserted to a single, fully integrated instrument thatcontains the following subsystems (FIG. 59) as described in U.S. patentapplication Ser. No. 12/396,110, entitled Ruggedized Apparatus forAnalysis of Nucleic Acid and Proteins, Published as 2009/0229983 (See,e.g., para. 65-76); U.S. patent application Ser. No. 12/080,746 entitledMethods for Rapid Multiplexed Amplification of Target Nucleic Acids,Published as 2009/0023602 (See, e.g, para. 54 and 56-67): U.S. patentapplication Ser. No. 12/080,751 entitled Integrated Nucleic AcidAnalysis, Published as 2009/0059222 (See, e.g., para. 139-144); U.S.patent application Ser. No. 12/080,745, entitled Plastic MicrofluidicSeparation and Detection Platforms, Published as 2009/0020427 (See,e.g., para 95), all of which are incorporated herein in theirentireties, by reference. The subsystems include:

Pneumatic Subsystem

A pneumatic and vacuum pump is connected to a pneumatic manifold (FIG.44, 61 as described in Example 4) through a set of tanks and solenoidvalves. This manifold is in turn connected to the pneumatic ports of thebiochip through the pneumatic interface. The solenoid valves arecontrolled by the process controller.

Thermal Subsystem

A peltier based thermal cycling system is coupled to the thermal cyclingchambers (FIG. 35, 62) of the biochip to rapidly cycle the reactionswithin these channels. As noted in Example 4, a clamp applies pressureto the amplification region to allow efficient thermal transfer with thethermal cycler. The reaction temperatures and dwell times are controlledby the process controller.

High Voltage Subsystem

A high voltage power supply is coupled to the anode (FIG. 55, 63) andcathode (FIG. 55, 64) portions of the separation biochip. Note that theanode electrodes pass through the pneumatic and fluidic pates at anodeportion (FIG. 44, 65 and FIG. 35, 65) and the cathode electrode isincorporated into the cathode portion within the fluidic plate andpneumatic plates (FIG. 44, 66 and FIG. 35, 66) to gain access to theseparation and detection biochip. Application of a high voltagegenerates an electric field along the channels to effectpre-electrophoresis, sample injection, and separation and detection. Thehigh voltage subsystem is controlled by the process controller.

Optical Subsystem

An optical system is coupled to the excitation and detection window ofseparation and detection biochip that is within the biochip (FIGS. 55,67 and 56, 67). Note that the light passes through an open window on thepneumatic and fluidic plates (refer to FIG. 44, 38 and FIG. 35, 37). Theoptical system consists of a laser that is used to induce fluorescencefrom labeled DNA fragments within the separation channel. A set ofdichroic mirrors is used to separate wavelength components of thefluorescence. A set of photomultiplier tubes are used to detect thefluorescence signals. A set of optical components is used to transferlight (laser excitation and emitted fluorescence) between the laser andthe separation and detection biochip, and the separation and detectionbiochip and the detectors. The optical subsystem is controlled by theprocess controller.

Process Controller

A computer based controller that can accept a pre-defined process scriptand implement the script automatically by reading the script andcontrolling the subsystems accordingly. Note that the computer alsoperforms data processing and data analysis as required.

The sample-in to results out process was performed as follows. Fiveswabs were collected from donors. The swabs were collected by pressingthe head of a Bode Secur-swab swab to the inside of the cheek asinstructed by the manufacturer. Each of the 5 swabs was inserted intoone of the swab chambers of the biochip, the biochip was placed into theinstrument, and the automated process script was initiated to carry outthe swab-in to results-out analysis. An automated script with thefollowing steps was executed:

-   -   Initialization. All valves on the biochip were closed by        applying pressure on valve lines. The valves in Example 5 were        actuated with 20 psig of pressure to close and vented to        atmosphere to open. The exception is valve V3 (FIG. 39, 41)        which was maintained in the open position by the application of        a vacuum of −11 psig. For simplicity, valve numbers are referred        to based on their position relative to the pneumatic plate (even        though the valves are actually located within the valve        assembly).    -   Lysis. 550 microliters of lysis solution was pneumatically        loaded from the lysis reagent chamber (FIG. 39, 20) into the        swab chamber (FIG. 39, 19) by opening valve V1 (FIG. 39, 39) and        applying a pressure of 3 psig to drive line DL1 (FIG. 39, 68)        for 30 seconds and then 5.5 psig for 60 sec. Valve V1 (FIG. 39,        39) was closed and drive line 1 (FIG. 39, 68) was deactivated.        550 microliters of ethanol from the ethanol reagent chamber        (FIG. 39, 21) was pneumatically loaded into the swab chamber        (FIG. 39, 19) by opening valve V2 (FIG. 39, 40) and applying a        drive pressure of 5.5 psig to drive line 2 (FIG. 39, 69) for 30        seconds.    -   Chaotic bubbling. Air was pneumatically driven into the swab        chamber (FIG. 39, 19) by opening valve V2 (FIG. 39, 40) and        applying a pneumatic pressure of 5.5 psig to drive line 2 (FIG.        39, 69) for 30 seconds. Valve 2 (FIG. 39, 40) was closed and        drive line 2 (FIG. 39, 69) was deactivated. The air that was        driven into the swab chamber bubbled through the lysis solution        chaotically agitating both the lysis reagent and the swab head.        This lysed the cells and released DNA.    -   Queuing. The lysate was pulled from the swab chamber (FIG. 39,        19) through a particulate filter (FIG. 39, 11) into a holding        chamber (FIG. 39, 24) by maintaining valve V3 (FIG. 39, 41) in        the open position while applying a vacuum of −7 psig to drive        line 3 (FIG. 39, 70) for 30 seconds to transfer the lysate.        Valve 3 (FIG. 39, 41) was closed and drive line 3 (FIG. 39, 70)        was deactivated.    -   DNA binding. Lysate from the holding chamber (FIG. 39, 24) was        pneumatically driven through the purification filter (FIG. 39,        12) and into the swab chamber (FIG. 39, 19) by opening valve V4        (FIG. 39, 42) and a set of valves V5 (FIG. 39, 43) and applying        a pressure of 5 psig for 60 sec to drive line DL3 (FIG. 39, 70).        Valves V4 (FIG. 39, 42) and V5 (FIG. 39, 43) were closed and DL3        (FIG. 39, 70) was deactivated. DNA within the lysate bound to        the purification filter (FIG. 39, 12). Note that the swab        chamber also served as a waste chamber following generation and        processing of the lysate; this dual use eliminated the need for        another large volume chamber on the macrofluidic block. Separate        waste chambers from each sample can optionally be included if        retention of lysate is desired.    -   Wash. Wash solution was pneumatically driven from the wash        reagent chamber (FIG. 39, 22) through the purification filter        (FIG. 39, 12) into the swab chamber (FIG. 39, 19) by opening        valves V5 (FIG. 39, 43) and V6 (FIG. 39, 46), and applying a        pneumatic pressure of 13 psig to drive line DL4 (FIG. 39, 71)        for 90 seconds. Valves V5 (FIG. 39, 43) and V6 (FIG. 39, 46)        were closed and DL4 (FIG. 39, 71) deactivated. 3 ml of wash was        passed through the purification filter to remove contaminants        and particulate debris from the bound DNA. Two additional washes        allowed cleaning of adjacent channels.    -   Dry. Air was pneumatically driven through the purification        filter (FIG. 39, 12) and into the swab chamber (FIG. 39, 19) by        opening valve V5 (FIG. 39, 43) and V6 (FIG. 39, 46), and        applying a pressure of 13 psig to drive line DL4 (FIG. 39, 46)        for 185 seconds. Valves V5 and V6 were closed and DL4 (FIG. 39,        71) was deactivated. The air partially to fully dries the        purification filter.    -   Elution. Elution buffer is pneumatically driven from the elution        reagent chamber (FIG. 39, 23) through the purification filter        (FIG. 39, 12) and into an eluate holding chamber (FIG. 39, 25)        by opening valves V6 (FIG. 39, 46), V7 (FIG. 39, 45) and V8        (FIG. 39, 47) and applying a pressure of 5 psig to drive line        DL5 (FIG. 39, 72) for 120 seconds. Valves V6 (FIG. 39, 46), V7        (FIG. 39, 45) and V8 (FIG. 39, 47) were closed and DL5 (FIG. 39,        72) was deactivated. 300 microliters of elution buffer was        passed through the purification filter (FIG. 39, 12) to release        purified DNA that was bound to the purification filter.    -   Homogenization. Air was pneumatically driven into the eluate        holding chamber by opening valve V6 (FIG. 39, 46), and V8 (FIG.        39, 47) and applying a pressure of 5 psig to drive line DL4        (FIG. 39, 71) for 60 seconds. Valves V6 (FIG. 39, 46), and V8        (FIG. 39, 47) were and DL4 (FIG. 39, 71) was deactivated. The        air that was driven into the eluate holding chamber bubbles        through the eluate to agitation and homogenize the DNA within        the eluate. Optionally, a portion of the eluate can be routed to        a storage chamber or onto a storage filter if a retention sample        of the purified DNA is desired.    -   Eluate metering. Eluate was pneumatically driven from the Eluate        holding chamber (FIG. 39, 25) into the eluate metering chambers        (FIG. 40, 8) by opening Valve V10 (FIG. 39, 48) and applying        pressure to DL6 (FIG. 39, 73) 1 psig for 40 seconds. Valves V10        (FIG. 39, 48) was closed and DL6 (FIG. 39, 73) was deactivated.        Each eluate filled the metering chamber and stopped at a vent        membrane. Excess eluate was pneumatically driven back into the        Eluate holding chamber by opening valves V10 (FIG. 39, 48) and        V11 (FIG. 40, 50) and applying a pressure of 2 psig to Drive        line DL7 (FIG. 40, 49) for 25 seconds. Valves V10 (FIG. 39, 48)        and V11 (FIG. 40, 50) were closed and DL7 (FIG. 40, 49) was        deactivated.    -   Reconstitute PCR cake. The eluate was pneumatically driven from        the eluate metering chamber (FIG. 40, 8) into the PCR cake        chamber (FIG. 40, 10) by opening valves V11 (FIG. 40, 50) and        V12 (FIG. 40, 51) and applying a drive sequence of 0.2 psig for        30 seconds, then 0.4 psig for 30 sec, then 0.6 psig for 60 sec        to drive line DL7 (FIG. 41, 49). Valves V11 (FIG. 40, 50) and        V12 (FIG. 40, 51) were closed and (FIG. 41, 49) was deactivated.        20.5 microliters of metered eluate was transferred to        reconstitute the cake containing the lyophilized PCR reaction        mix to generate the PCR mix for amplification.    -   Transfer into thermal cycling chamber. PCR reaction mix was        pneumatically driven from the cake chamber (FIG. 40, 10) into        the thermal cycling chambers (FIG. 40, 62) by opening valves V11        (FIG. 40, 50), V12 (FIG. 40, 51), V13 (FIG. 40, 52), and V14        (FIG. 40, 53), and applying a sequentially increasing drive        pressure sequence of 0.2 psig for 30 seconds, then 0.4 psig for        30 sec, then 0.6 psig for 30 sec to drive line DL7 (FIG. 40,        49). Valves V11 (FIG. 40, 50), V12 (FIG. 40, 51), V13 (FIG. 40,        52), and V14 (FIG. 40, 53) were closed, and DL7 (FIG. 40, 49)        was deactivated. The PCR reaction mix was transferred into the        thermal cycling chambers and stops at the queuing vent membrane.    -   Thermal cycle. A thirty-one cycle protocol was applied to cycle        the reaction within the thermal cycling chambers (FIG. 40, 62)        to generate labeled amplicons. (See, Giese, H., et al. (2009).        “Fast multiplexed polymerase chain reaction for conventional and        microfluidic short tandem repeat analysis.” J Forensic Sci        54(6): 1287-96, and application Ser. No. 12/080,746, published        as 2009/0023603, entitled “Methods for Rapid Multiplexed        Amplification of Target Nucleic Acids,” both of which are        incorporated herein by reference), The cycling conditions were        as follows: Hotstart 93° C.×20 seconds followed by 31 cycles of        (93° C.×4 seconds, 56° C.×15 seconds, and 70° C.×7 seconds)        followed by a final extension of 70° C.×90 seconds.    -   Meter PCR product. PCR product was pneumatically driven from the        thermal cycling chambers (FIG. 40, 62) into the PCR metering        chamber (FIG. 41, 74) by opening valves V11 (FIG. 40, 50), V12        (FIG. 40, 51), V13 (FIG. 40, 52), V14 (FIG. 40, 53), and V30        (FIG. 41, 111) and applying a sequentially increasing drive        pressure sequence of 0.2 psig for 30 seconds, then 0.4 psig for        30 sec, then 0.6 psig for 45 sec drive line DL7 (FIG. 40, 49).        Valves V11 (FIG. 40, 50), V12 (FIG. 40, 51), V13 (FIG. 40, 52),        V14 (FIG. 40, 53) and V30 (FIG. 41, 111) were closed and DL7        (FIG. 40, 49) was deactivated. The PCR product flows into the        metering chamber and stops at a vent membrane.    -   Meter formamide. Formamide was pneumatically driven from the        formamide reagent chamber (FIG. 41, 60) into the formamide        metering chamber (FIG. 41, 76) by applying a pressure of 1 psig        for 50 seconds to drive line DL8 (FIG. 41, 75). Drive line DL8        (FIG. 41, 75) was deactivated. In this step, a 6^(th) volume of        formamide was metered (FIG. 41, 77) for reconstituting a cake to        generate a control sample. The formamide flows into the metering        chamber and stops at a vent membrane. Excess formamide is        pneumatically driven from the formamide chamber into a waste        chamber by opening valve V15 (FIG. 41, 54) and applying a        pressure of 3 psig for 180 seconds to drive line DL8 (FIG. 41,        75). Valve V15 (FIG. 41, 54) was closed and DL8 (FIG. 41, 75)        was deactivated. All of the excess formamide reagent chamber and        drive line was transferred into the waste chamber.    -   Join PCR product and formamide. Metered PCR product was        pneumatically driven from the PCR metering chamber (FIG. 41, 74)        into the joining chamber (FIG. 41, 78) by opening valve V18        (FIG. 41, 57), V17 (FIG. 41, 80) and V20 (FIG. 41, 81) and        applying a 4 step drive profile of .0.2, 0.3, 0.4, and 0.5 psig        for 30, 30, 30, and 30 sec respectively to drive line DL9 (FIG.        41, 79). Valve V18 (FIG. 41, 57), V17 (FIG. 41, 80) and V20        (FIG. 41, 81) were closed and DL9 (FIG. 41, 79) was deactivated.        Metered formamide was pneumatically driven from the formamide        metering chamber (FIG. 41, 76) into the joining chamber (FIG.        41, 78) by opening valve V16 (FIG. 41, 55) and V20 (FIG. 41, 81)        and applying an increasing pressure of 0.2, 0.4 and 0.6 psig for        30, 30, and 60 seconds to drive line DL8 (FIG. 41, 75). Valve        V16 (FIG. 41, 55) and V20 (FIG. 41, 81) were closed and DL8        (FIG. 41, 75) was deactivated. The joining chamber allows the        metered PCR and the metered formamide which originate from two        independent flow to be combined to form the sample for        separation and detection.    -   Reconstitute ILS cake. The sample for separation and detection        was pneumatically driven into the ILS cake chambers (FIG. 41,        35) by opening valves V16 (FIG. 41, 55) and V19 (FIG. 41, 56),        and applying an increasing pressure of 0.2, 0.4 and 0.6 psig for        30, 30, and 60 seconds to drive line DL8 (FIG. 41, 75). V16        (FIG. 41, 55) and V19 (FIG. 41, 56) were closed and DL8 (FIG.        41, 75) was deactivated. The sample for separation and detection        which is composed of 4.1 microliters of PCR product and 16.4        microliters of formamide reconstituted the ILS cake within the        chamber to generate the separation and detection sample.    -   Reconstitute Control+ILS cake. The 6^(th) metered formamide        volume is pneumatically driven into the control+ILS cake chamber        (FIG. 41, 83) by opening valves V16 and V20, and applying an        increasing pressure of 0.2, 0.4 and 0.6 psig for 30, 30, and 60        seconds to drive line DL9. V16 and V20 are closed and DL9 is        deactivated. The 20.5 microliters of formamide reconstituted the        cake within the chamber to generate the separation and detection        sample.    -   Inject samples into separation channel. The 6 separation and        detection samples were pneumatically driven from the cake        chambers (FIGS. 41, 35 and 82) through a debubbling chamber        (FIG. 41, 83) to fill the cathode chamber (FIG. 41, 84) and a        sample waste chamber (FIG. 41, 85) by opening valve V21 (FIG.        41, 86) and V22 (FIG. 41, 87) and applying a pressure to drive        line DL8 (FIG. 41, 74) with a sequentially stepped profile of        0.4, 0.6, 1.0, 1.5 and 2.0 psig for 30, 30, 30, 30, and 30        seconds respectively. (FIG. 41, 86) and V22 (FIG. 41, 87) were        closed and DL8 (FIG. 41, 74) was deactivated. The DNA within the        sample was injected from the cathode chamber (FIG. 41, 84) into        the separation portion of the biochip (FIG. 56 and FIG. 55, 88),        by applying a voltage of 4400 V was applied to bias the cathode        (FIGS. 55 and 56, 63) and anode (FIGS. 55 and 56, 64) for 35        seconds.    -   Separate and detect DNA. TTE was pneumatically driven from the        from the TTE reagent reservoir (FIG. 41, 59) to fill the cathode        (FIG. 41, 84) and to fill TTE waste chambers (FIGS. 41, 92, 93,        and 13) by opening valves V25 (FIG. 41, 91), V24 (FIG. 41, 90),        V26 (FIG. 41, 95), V27 (FIG. 41, 96), and V28 (FIG. 41, 97) and        applying a pressure to drive line DL10 (FIG. 41, 98) for 2 psig        for 240 seconds. valves V25 (FIG. 41, 91), V24 (FIG. 41, 90),        V26 (FIG. 41, 95), V27 (FIG. 41, 96), and V28 (FIG. 41, 97) were        closed and DL10 (FIG. 41,98) was deactivated. The flow of TTE        through the cathodes displaced the sample within the cathodes.        DNA that was injected into the separation portion of the S&D        biochip (FIG. 55) traveled down the separation portion of the        biochip (FIG. 56, 88), when a 6400 V was applied to bias the        cathode (FIG. 55, 63) and anode (FIG. 55, 64) for 30 min. The        optical system was also activated to effect laser induced        fluorescence excitation and detection at the excitation and        detection window. The laser was set to 200 mW and the data        collection rate of 5 Hz is implemented. The fluorescent signal        travels through the detection path to the photomultiplier tubes.        There, the fluorescence is converted into a signal that is        recorded by the system software.

Electropherograms (FIGS. 60 and 61) were generated following thescripted process steps as outlined above. The control electropherogram(FIG. 60) was generated for the control ILS sample (fragmentsfluorescently labeled with ROX). The x-axis represents data collectioncounts, with each count indicating the time at which the labeledfragment arrive at the detection zone. Several of the size standards areindicated by arrows, and lower molecular weight fragments migrate morerapidly than higher molecular weight fragments and are detected earlier(i.e. to the left of the graph). The Y-axis shows the relativefluorescent units (rfu) for each peak. FIG. 61 shows the sizes of theSTR fragments generated for one buccal swab sample. Each of fluorescentdyes used to label the STR primers in the PCR reaction mix and the ILSare shown in individual panels. Fluorescein-labeled fragments are shownin the top panel, JOE-labeled fragments are shown in the second panelfrom the top, TAMRA-labeled fragments are shown in the third panel fromthe top, and ROX-labeled ILS fragments are shown in the bottom panel.The x- and y-axes are the same as described for FIG. 60. The entireprocess from insertion of samples to generation of the electropherogramrequired approximately 90 minutes and approximately 215 scripted processsteps. Many process steps can be shortened, and the process can beperformed in less than 45 minutes if desired.

Example 6 Injection Molding of a Fully Integrated Biochip that PurifiesNucleic Acids, Amplifies the Purified DNA, Electrophoretically Separatesthe Amplified DNA and Generates an STR Profile Using an Automated Script

This biochip resembles that of Example 5, with the fluidic and pneumaticplates fabricated by injection molding. The stationary, unitary plasticbiochip that accepts 5 buccal swabs and generates STR profiles consistsof the following parts:

Fluidic Subassembly

This subassembly transfers and processes fluids within the biochip,interacting with the pneumatic subassembly, macrofluidic processingsubassembly, valve subassembly, and separation and detectionsubassembly. It was fabricated by injection molding of COP with aninterconnected set of channels, chambers, and membrane and filterfeatures on both the top and bottom sides of the thermoplastic sheet.FIG. 62 shows the top side of the fluidic plate 601, FIG. 63 shows thebottom side of the fluidic plate 601, and FIG. 64 shows a transparentview of the fluidic plate 601, showing features from both sides. FIG. 65shows a photo of the injection molded fluidic plate 601. The injectionmolded fluidic plate has dimensions of 276 mm×117 mm×2.5 mm. Both thetop and bottom sides of the plate are covered with patterned thinplastic films. FIG. 66 shows the top patterned thin film 602 and FIG. 67shows the bottom patterned thin film 603.

The flow path for a single sample within the injection molded fluidicplate is shown in FIG. 68. The sample flows path 4 passes through apurification section (FIG. 68, 5), PCR section (FIG. 68, 6) andseparation and detection section (FIG. 68, 7). Expanded views of thepurification section, PCR section and separation and detection sectionsare shown in FIGS. 69, 70, and 71 respectively. The injection moldedfluidic plate has a particulate filter region (FIG. 69, 11), apurification membrane region (FIG. 69, 12), and several types ofchambers, including metering chambers (FIG. 69, 8), reconstitutionchambers (FIG. 69, 10), joining chambers (FIG. 70, 78), waste chambers(FIGS. 70, 92, 93, and 94), and cathodes (FIG. 71, 84) and an anode(FIG. 64, 65).

The top and bottom sides of the fluidic plate were covered withpatterned thin plastic films and attached by solvent bonding. Bonding ofthe patterned thin films to the fluidic plate can also be performedultrasonically, thermally, and using adhesives. Features on the plateand films can be added to facilitate the bonding method; for example,energy director ridges can be placed at the sites of ultrasonic welding.The thin films have a thickness of 100 microns and were fabricated byCNC machining; optionally, they can be fabricated by die cutting, orlaser cutting. The features in the thin plastic films include throughholes and were aligned to the corresponding features on the injectionmolded layer to provide access to the fluidic sandwich layer.Purification membranes (FIG. 69, 12), particulate filters (FIG. 69, 11)and vent membranes were attached to this subassembly by thermal weldingprior to bonding of the thin films. Similarly, these membranes andfilters can be attached by ultrasonic welding and heat staking

Pneumatic Subassembly

This subassembly couples the pneumatic drive of the instrument tofluidic subassembly (as described in Example 4) to pneumatically drivefluids within the fluidic subassembly and to pneumatically activatingvalves within the biochip. This subassembly was fabricated by injectionmolding of COP with an interconnected set of channels, chambers, andmembrane and filter features on both the top and bottom sides of thethermoplastic sheet. FIG. 72 shows the top side of the pneumatic plate,FIG. 73 shows the bottom side of the pneumatic plate, and FIG. 74 showsa transparent view of the pneumatic plate, showing features from bothsides. FIG. 75 shows a photograph of the injection molded pneumaticplate. The injection molded pneumatic plate has dimensions of 277.6mm×117.7 mm×2.50 mm. Following bonding, all pneumatic subassemblyfeatures align with the fluidic subassembly features. The top side ofthe plate is covered with patterned thin plastic film with through holesto allow access to the plate. FIG. 76 shows the top patterned thin film.The bottom side of the plate is covered when the patterned thin filmrepresenting the valve subassembly is attached by solvent bonding; thethin film is patterned by CNC machining. The thin films have a thicknessof 100 microns. Patterns can be cut prior to bonding or followingbonding. Prior to bonding the top and bottom thin films to the pneumaticplate, vent membranes are incorporated (FIG. 68, 17). Attachment of thethin films to the pneumatic plate was accomplished with solvents but canalso be performed ultrasonically, thermally, and using adhesives.

The macrofluidic processing subassembly and separation and detectionsubassembly are the same as described in Example 5. In this biochip, thevalve assembly is a thin thermoplastic film that is 40 microns, 50microns, or 100 microns thick. The pneumatically actuated rigid valvelayer (described in Example 2) separates the pneumatic and the fluidicassemblies. When pneumatically activated, this layer deflects to controlflow of fluids (including air) within the fluidic layer.

The pneumatic-valve-fluidic stack subassembly was fabricated by solventbonding the pneumatic assembly to the fluidic assembly. The chambers andchannels on the bottom side of the pneumatic plate are sealed whensolvent bonded to the fluidic layer. The macrofluidic block was attachedto the cover by clamping using a gasket and screws. The block wasattached to the pneumatic subassembly using double-sided PSA tape.Alternatively, the block attachments can be made by solvent and thermalbonding. Finally, the separation and detection subassembly was attachedto the fluidic subassembly using PSA tape, and the subassemblies wereoriented such that nucleic acid flow in within the separation anddetection biochip was conducted in the opposite direction as NApurification and amplification.

To test the biochip, liquid and lyophilized reagents are loaded asdescribed in Example 5 and the biochip is inserted into the fullyintegrated instrument of Example 5. Five buccal swabs are collected fromdonors, and each of the 5 swabs was inserted one of the swab chambers ofthe biochip. An automated script with the following steps is executed:

-   -   Initialization. All valves on the biochip are closed by applying        pressure of 75 psig on all valve lines. The exception is valve        V3 which was maintained in the open position by the application        of a vacuum of −11 psig. For simplicity, valve numbers are        referred to based on their position relative to the pneumatic        plate (even though the valves are actually located within the        valve assembly).    -   Lysis. 550 microliters of lysis solution is pneumatically loaded        from the lysis reagent chamber (FIG. 69, 20) into the swab        chamber (FIG. 69, 19) by opening valve V1 (FIG. 69, 39) and        applying a pressure of 3 psig to drive line DL1 (FIG. 69, 68)        for 30 seconds and then 5.5 psig for 60 sec. Valve V1 (FIG. 69,        39) is closed and drive line 1 (FIG. 69, 68) is deactivated. 550        microliters of ethanol from the ethanol reagent chamber (FIG.        69, 21) is pneumatically loaded into the swab chamber (FIG. 69,        19) by opening valve V2 (FIG. 69, 40) and applying a drive        pressure of 5.5 psig to drive line 2 (FIG. 69, 69) for 30        seconds.    -   Chaotic bubbling. Air is pneumatically driven into the swab        chamber (FIG. 69, 19) by opening valve V2 (FIG. 69, 40) and        applying a pneumatic pressure of 5.5 psig to drive line 2 (FIG.        69, 69) for 30 seconds. Valve 2 (FIG. 69, 40) is closed and        drive line 2 (FIG. 69, 69) is deactivated. The air that is        driven into the swab chamber bubbles through the lysis solution        chaotically agitating both the lysis reagent and the swab head.        This lysed the cells and released DNA.    -   Queuing. The lysate is pulled from the swab chamber (FIG. 69,        19) through a particulate filter (FIG. 69, 11) into a holding        chamber (FIG. 69, 24) by maintaining valve V3 (FIG. 69, 41) in        the open position while applying a vacuum of −7 psig to drive        line 3 (FIG. 69, 70) for 30 seconds. Valve 3 (FIG. 69, 41) is        closed and drive line 3 (FIG. 69, 70) is deactivated.    -   DNA binding. Lysate from the holding chamber (FIG. 69, 24) is        pneumatically driven through the purification filter (FIG. 69,        12) and into the swab chamber (FIG. 69, 19) by opening valve V4        (FIG. 69, 42) and a set of valves V5 (FIG. 69, 43) and applying        a pressure of 5 psig for 60 sec to drive line DL3 (FIG. 69, 70).        Valves V4 (FIG. 69, 42) and V5 (FIG. 69, 43) were closed and DL3        (FIG. 69, 70) is deactivated. DNA within the lysate binds to the        purification filter (FIG. 69, 12). Note that the swab chamber        also serves as a waste chamber following generation and        processing of the lysate; this dual use eliminates the need for        another large volume chamber on the macrofluidic block. Separate        waste chambers from each sample can optionally be included if        retention of lysate is desired.    -   Wash. Wash solution is pneumatically driven from the wash        solution chamber (FIG. 69, 22) through the purification filter        (FIG. 69, 12) into the swab chamber (FIG. 69, 19) by opening        valves V5 (FIG. 69, 43) and V6 (FIG. 69, 46), and applying a        pneumatic pressure of 13 psig to drive line DL4 (FIG. 69, 71)        for 90 seconds. Valves V5 (FIG. 69, 43) and V6 (FIG. 69, 46)        were closed and DL4 (FIG. 69, 71) deactivated. 3 ml of wash is        passed through the purification filter to remove contaminants        and particulate debris from the bound DNA. Two additional washes        allow cleaning of adjacent channels.    -   Dry. Air is pneumatically driven through the purification filter        (FIG. 69, 12) and into the swab chamber (FIG. 69, 19) by opening        valve V5 (FIG. 69, 43) and V6 (FIG. 69, 46), and applying a        pressure of 13 psig to drive line DL4 (FIG. 69, 46) for 185        seconds. Valves V5 and V6 are closed and DL4 (FIG. 69, 71) is        deactivated. The air partially to fully dries the purification        filter.    -   Elution. Elution buffer is pneumatically driven from the elution        reagent chamber (FIG. 69, 23) through the purification filter        (FIG. 69, 12) and into an eluate holding chamber (FIG. 69, 25)        by opening valves V6 (FIG. 69, 46), V7 (FIG. 69, 45) and V8        (FIG. 69, 47) and applying a pressure of 5 psig to drive line        DL5 (FIG. 69, 72) for 120 seconds. Valves V6 (FIG. 69, 46), V7        (FIG. 69, 45) and V8 (FIG. 69, 47) are closed and DL5 (FIG. 69,        72) was deactivated. 300 microliters of elution buffer is passed        through the purification filter (FIG. 69, 12) to releases        purified DNA that was bound to the purification filter.        Optionally, a portion of the eluate can be routed to a filter        for storage as a retention sample. In this case, the filter        would be incorporated into a mechanism that would allow it to be        removed from the biochip.    -   Homogenization. Air is pneumatically driven into the eluate        holding chamber by opening valve V6 (FIG. 69, 46), and V8 (FIG.        69, 47) and applying a pressure of 5 psig to drive line DL4        (FIG. 69, 71) for 60 seconds. Valves V6 (FIG. 69, 46), and V8        (FIG. 69, 47) were and DL4 (FIG. 69, 71) is deactivated. The air        that is driven into the eluate holding chamber bubbles through        the eluate to agitation and homogenize the DNA within the        eluate.    -   Eluate metering. Eluate is pneumatically driven from the Eluate        holding chamber (FIG. 69, 25) into the eluate metering chambers        (FIG. 70, 8) by opening Valve V10 (FIG. 69, 48) and applying        pressure to DL6 (FIG. 69, 73) 1 psig for 40 seconds. Valves V10        (FIG. 69, 48) is closed and DL6 (FIG. 69, 73) is deactivated.        Each eluate fills the metering chamber and stopped at a vent        membrane. Excess eluate is pneumatically driven back into the        Eluate holding chamber by opening valves V10 (FIG. 69, 48) and        V11 (FIG. 70, 50) and applying a pressure of 2 psig to Drive        line DL7 (FIG. 70, 49) for 25 seconds. Valves V10 (FIG. 69, 48)        and V11 (FIG. 70, 50) were closed and DL7 (FIG. 70, 49) is        deactivated.    -   Reconstitute PCR cake. The eluate is pneumatically driven from        the eluate metering chamber (FIG. 70, 8) into the PCR cake        reconstitution and reciprocation chamber (FIG. 70, 610) by        opening valve V11 (FIG. 70, V11) and linearly increasing the        drive pressure of DL7 (FIG. 71, 49) from 0 to 15 psig over 15        seconds. The chamber of (FIG. 70, 610) is designed to hold the        lyophilized PCR cake, allow reconstitution of the PCR cake, and        reciprocal mixing of the PCR reaction. The volume of the chamber        is approximately 2.2 times that of the eluate which is defined        by the eluate metering chamber (FIG. 70, 8). The PCR cake is        placed and located at the input side of the chamber. The output        of the chamber is sealed by a valve. The air between the eluate        and the valve V13 (FIG. 71, 52) is compressed allowing the        eluate to move within the chamber and come in contact with the        lyophilized PCR cake. The cake is allowed to reconstitute by        maintaining the pressure of DL7 at 15 psig for 60 seconds. 11.5        microliters of metered eluate is transferred to reconstitute the        cake containing the lyophilized PCR reaction mix to generate the        PCR mix for amplification.    -   Reciprocally Mix the PCR solution—The PCR solution (i.e. PCR        cake that is reconstituted with eluate) is then moved from the        PCR reconstitution and reciprocation chamber (FIG. 70, 10) back        to the eluate metering chamber (FIG. 70, 8) by linearly        decreasing the pressure of drive line DL7 from 15 psig to 0 psig        over 15 seconds. The air between the eluate and valve V13 (FIG.        71, 52) is compressed and acts as an air spring to push against        the PCR mix moving it towards the metering chamber (FIG. 71, 8).        The PCR mix is reciprocally mixed by linearly increasing the        drive pressure of DL7 from 0 psig to 15 psig over 15 seconds,        and then from 15 psig to 0 psig over 15 seconds. Valve V12 (FIG.        70, 49) is closed and drive line DL7 (FIG. 70, 49) is        deactivated    -   Transfer into thermal cycling chamber. PCR reaction mix is        pneumatically driven from the eluate metering chamber (FIG. 70,        8) into the thermal cycling chambers (FIG. 70, 62) by opening        valves V11 (FIG. 70, 50), V13 (FIG. 70, 52), and V14 (FIG. 70,        53), and applying a sequentially increasing drive pressure        sequence of 0.2 psig for 30 seconds, then 0.4 psig for 30 sec,        then 0.6 psig for 30 sec to drive line DL7 (FIG. 70, 49). Valves        V12 (FIG. 70, 51) and V14 (FIG. 70, 53) were closed, and DL7        (FIG. 70, 49) is deactivated. The PCR reaction mix is        transferred into the thermal cycling chambers and stops at the        queuing vent membrane.    -   Thermal cycle. A thirty-one cycle protocol is applied to cycle        the reaction within the thermal cycling chambers (FIG. 70, 62)        to generate labeled amplicons. The cycling conditions are as        follows: Hotstart 93° C.×20 seconds followed by 31 cycles of        (93° C.×4 seconds, 56° C.×15 seconds, and 70° C.×7 seconds)        followed by a final extension of 70° C.×90 seconds.    -   Meter PCR product. PCR product is pneumatically driven from the        thermal cycling chambers (FIG. 70, 62) into the PCR metering        chamber (FIG. 71, 74) by opening valves V11 (FIG. 70, 50), V13        (FIG. 70, 52) and V14 (FIG. 70, 53) and V30 (FIG. 71, 111), and        applying a sequentially increasing drive pressure sequence of        0.2 psig for 30 seconds, then 0.4 psig for 30 sec, then 0.6 psig        for 45 sec drive line DL7 (FIG. 70, 49). Valves V11 (FIG. 70,        50), V13 (FIG. 70, 52), V14 (FIG. 70, 53), and V30 (FIG. 71,        111) were closed and DL7 (FIG. 70, 49) is deactivated. The PCR        product flows into the metering chamber and stops at a vent        membrane.    -   Meter formamide. Formamide is pneumatically driven from the        formamide reagent chamber (FIG. 71, 60) into the formamide        metering chamber (FIG. 71, 76) by applying a pressure of 1 psig        for 50 seconds to drive line DL8 (FIG. 71, 75). Drive line DL8        (FIG. 71, 75) is deactivated. In this step, a 6^(th) volume of        formamide is metered (FIG. 71, 77) for reconstituting a cake to        generate a control sample. The formamide flows into the metering        chamber and stops at a vent membrane. Excess formamide is        pneumatically driven from the formamide chamber into a waste        chamber by opening valve V15 (FIG. 71, 54) and applying a        pressure of 3 psig for 180 seconds to drive line DL8 (FIG. 71,        75). Valve V15 (FIG. 71, 54) is closed and DL8 (FIG. 71, 75) is        deactivated. All of the excess formamide reagent chamber and        drive line is transferred into the waste chamber.    -   Join PCR product and formamide. Metered PCR product is        pneumatically driven from the PCR metering chamber (FIG. 71, 74)        into the joining chamber (FIG. 71, 78) by opening valve V18        (FIG. 71, 57), V17 (FIG. 71, 80) and V20 (FIG. 71,81) and        applying a 4 step drive profile of .0.2, 0.3, 0.4, and 0.5 psig        for 30, 30, 30, and 30 sec respectively to drive line DL9 (FIG.        71, 79). Valve V18 (FIG. 71, 57), V17 (FIG. 71, 80) and V20        (FIG. 71,81) were closed and DL9 (FIG. 71, 79) is deactivated.        Metered formamide is pneumatically driven from the formamide        metering chamber (FIG. 71, 76) into the joining chamber (FIG.        71, 78) by V20 (FIG. 71, 81) by applying an increasing pressure        of 0.2, 0.4 and 0.6 psig for 30, 30, and 60 seconds to drive        line DL8 (FIG. 71, 75). Valve V20 (FIG. 71, 81) is closed and        DL8 (FIG. 71, 75) is deactivated. The joining chamber allows the        metered PCR and the metered formamide which originate from two        independent flow to be combined to form the sample for        separation and detection.    -   Reconstitute ILS cake. The sample for separation and detection        is pneumatically driven from the joining chamber (FIG. 71, 78)        into the ILS cake chambers (FIG. 71, 635) and ILS+Control cake        chamber (FIG. 71, 682) by opening valves V20 (FIG. 71, 81) and        linearly increasing the drive pressure of DL8 (FIG. 71, 75) from        0 to 15 psig over 15 seconds. The air between the sample and the        valve V21 (FIG. 71, 86) is compressed allowing the sample to        move within the chamber and come in contact with the lyophilized        ILS cake. The cake is allowed to reconstitute by maintaining the        pressure of DL8 (FIG. 71, 75) at 15 psig for 60 seconds. 11.5        microliters of metered sample is transferred to reconstitute the        cake containing the lyophilized internal lane standard to        generate the separation and detection sample.    -   Reciprocally Mix the separation and detection solution—The PCR        solution (i.e. ILS cake that is reconstituted with metered        formamide and metered PCR product) is then moved from the ILS        reconstitution and reciprocation chamber (FIG. 71, 635) back to        the joining chamber (FIG. 71, 78) by linearly decreasing the        pressure of drive line DL8 (FIG. 71, 75) from 15 psig to 0 psig        over 15 seconds. The air between the eluate and valve V13 (FIG.        71, 52) is compressed and will acts as an air spring to push        against the separation and detection sample to mix moving it        towards the formamide metering chamber (FIG. 71, 76). The        separation and detection sample is reciprocally mixed by        linearly increasing the drive pressure of DL8 (FIG. 71, 75) from        0 psig to 15 psig over 15 seconds, and then from 15 psig to 0        psig over 15 seconds. Drive line DL8 (FIG. 71, 75) was        deactivated.    -   Inject samples into separation channel. The 5 separation and        detection samples are pneumatically driven from the joining        chamber (FIG. 71, 78) and control sample from the formamide        metering chamber (FIG. 71, 76) through the ILS cake chamber        (FIGS. 71, 635, and 682), which now acts as a debubbling chamber        to fill the cathode chamber (FIG. 71, 84) and a sample waste        chamber (FIG. 71, 85) by opening valve V21 (FIG. 71, 86) and V22        (FIG. 71, 87) and applying a pressure to drive line DL9 (FIG.        71, 79) with a sequentially stepped profile of 0.4, 0.6, 1.0,        1.5 and 2.0 psig for 30, 30, 30, 30, and 30 seconds        respectively. Valves V21 (FIG. 71, 86) and V22 (FIG. 71, 87) are        closed and DL8 (FIG. 71, 74) is deactivated. The DNA within the        sample is injected from the cathode chamber (FIG. 71, 84) into        the separation portion of the biochip (FIG. 55 and FIG. 56, 88),        by applying a voltage of 4400 V is applied to bias the cathode        (FIGS. 55 and 56, 63) and anode (FIGS. 55 and 56, 64) for 35        seconds.    -   Separate and detect DNA. TTE is pneumatically driven from the        from the TTE reagent reservoir (FIG. 71, 59) to fill the cathode        (FIG. 71, 84) and to fill TTE waste chambers (FIGS. 71, 92, 93,        and 13) by opening valves V25 (FIG. 71, 91), V24 (FIG. 71, 90),        V26 (FIG. 71, 95), V27 (FIG. 71, 96), and V28 (FIG. 71, 97) and        applying a pressure to drive line DL10 (FIG. 71,98) for 2 psig        for 240 seconds. valves V25 (FIG. 71, 91), V24 (FIG. 71, 90),        V26 (FIG. 71, 95), V27 (FIG. 71, 96), and V28 (FIG. 71, 97) were        closed and DL10 (FIG. 71,98) is deactivated. The flow of TTE        through the cathodes displaces the sample within the cathodes.        DNA that is injected into the separation portion of the S&D        biochip (FIG. 55 or 5.24) travels injected down the separation        portion of the biochip, when a 6400 V is applied to bias the        cathode (FIGS. 55 and 56, 63) and anode (FIGS. 55 and 56, 64)        for 30 min. The optical system is also activated to effect laser        induced fluorescence excitation and detection at the excitation        and detection window. The laser is set to 200 mW and the data        collection rate of 5 Hz is implemented. The fluorescent signal        travels through the detection path to the photomultiplier tubes.        There, the fluorescence is converted into a signal that is        recorded by the system software.

Electropherograms are generated for the samples and control followingthe scripted process steps as outlined above. The entire process frominsertion of samples to generation of the electropherogram requiresapproximately 90 minutes. Many process steps can be shortened, and theprocess can be performed in less than 45 minutes if desired.

FIG. 77 illustrates the relationship of scripted processing steps andresultant processing steps for one portion of the process. Eluate ispneumatically driven from the Eluate holding chamber (FIG. 39, 25) toresult in a metered volume into the eluate metering chambers (FIG. 39,8) with the 10 step script of FIG. 77. The script steps consisting of 5valve and drive line state changes to move eluates from the holdingchambers to the metering chambers. The next 5 script steps remove theexcess eluate and push this volume into the holding chamber.Accordingly, the scripted processing steps (increasing and decreasingpressures on valves and drive lines) correspond to two resultantprocessing steps (the movement of sample from the eluate holding chamberto the eluate metering chamber and the movement of excess sample back tothe eluate holding chamber).

Example 7 Sample Splitting to Eliminate the Need for Nucleic AcidQuantitation

The amount of nucleic acids in a given sample, whether a forensic,clinical, or biothreat sample, is highly variable. In certain nucleicacid manipulations, reaction conditions require a particular range ofinput nucleic acid to be effective. In the laboratory, this problem isoften solved by performing a nucleic acid quantitation step prior to agiven manipulation. Nucleic acid quantitation has been developed formicrofluidic applications as described in patent application Ser. No.12/816,370 entitled “Improved Methods for Forensic DNA Quantitation,”incorporated herein by reference.

The instant invention provides another solution to the problem ofrequiring a certain range of nucleic acids in a microfluidic biochip.This approach does not involve quantitation but instead is based ondiluting a given sample one or more times to provide 2 or more nucleicacid concentrations for analysis, preferably in parallel. The inventionis exemplified using touch samples. Touch samples are forensic samplesthat consist of cells (primarily epithelial) that are left on surfacesafter exposure to the human body. They include fingerprints, skin cellsfound on clothing (e.g. a shirt collar), and oral epithelial cells foundon the opening of a soda can or the rim of a drinking glass. Thequantity of DNA that is recovered from touch DNA samples is highlyvariable. Touch samples generally contain up to 100 ng of DNA, and mosttouch samples contain from 0.5-10 ng DNA. Accordingly, the Touch SampleSystem will process swabs containing 0.5-100 ng DNA.

Although it is likely that most touch samples will contain less than 10ng of DNA, an unknown touch sample must be processed with theexpectation that the full 200-fold range of DNA must be processedcorrectly. In a manual amplification system, amplifying 0.5 ng would bereasonable but amplifying 100 ng would not. Accordingly, in manualsystems, DNA purified from touch samples is generally quantified priorto amplification. The microfluidic biochips of the invention allow touchsample DNA to be processed without quantitation by splitting purifiedDNA into two aliquots within the fluidic subassembly. One aliquot of theeluted DNA is amplified neat (assuming less than 10 ng DNA is present inthe amplification mix) and the other is diluted 20-fold (assuming 5-100ng DNA is present in the reaction mix). Both the neat and dilutedaliquots are amplified and separated and detected independently but inparallel. The neat sample allows effective amplification across a rangeof at least 0.05-10 ng of purified DNA, and the diluted sample allowseffective amplification across a range of at least 1-200 ng of purifiedDNA; the entire range of DNA content that cab be assayed effectivelyextends over a range of at least 40,000-fold.

Sample splitting microfluidics are incorporated into the biochips ofExamples 5 and 6 as follows. DNA is eluted in a volume of 20 microlitersusing the same DNA purification protocol. At this volume, the eluateholding chamber is removed from the macrofluidic processing subsystem,and a 20 microliter eluate holding chamber is placed on the fluidicplate of the fluidic subassembly. On that plate, the 20 microliters ofpurified DNA is routed to parallel paths:

-   -   In the neat path, 12 microliters of DNA is metered in a metering        chamber, transferred to the PCR cake reconstitution chamber, and        transferred to the PCR chambers for amplification. The chambers        will be expanded slightly to accommodate approximately 10        microliters of reconstituted reaction mix.    -   In the dilution path, approximately 2 microliters of DNA is        metered and joined with 38 μl of metered Elution Solution. The        diluted solution is then transferred to the cake reconstitution        chamber, and transferred to the PCR chambers for amplification.    -   Following amplification, the two solutions are processed in        parallel through separation and detection.

A flowchart of the sample splitting and dilution microfluidic circuit isshown in FIG. 78. Optionally, the purified DNA sample can be split intomore than two aliquots for subsequent processing, and the diluted samplecan be further diluted to generate additional aliquots for processing.For touch samples, such dilution is not necessary, but certain forensic,clinical diagnostic, and biothreat samples may contain much more DNA andtheir analysis facilitated by the processing of three or more aliquots.

Example 8 Plastic Background Fluorescence

The separation and detection biochips are fabricated in thermoplastic.Thermoplastics polymers when exposed to laser excitation autofluoresceto generate a background noise that is detected and processed by thedetection system. This autofluorescence degrades the signal-to-noiseratio of real signal peaks which raises the limit of detection of thesystem. Conventional S&D biochips for separation and detection arefabricated in glass or quartz substrates which exhibit lessautofluorescence per unit thickness compared to plastic substrates.Several considerations must be made when performing laser inducedfluorescence detection with plastic substrates:

-   (1) Selecting a plastic material that exhibits low    autofluorescence—Cyclin olefin copolymer (COC) and Cyclic olefin    polymer (COP) thermoplastic material is used to fabricate the    separation and detection biochips that are used in Examples 5 and 6.    These thermoplastics exhibit inherently lower autofluorescence in    the visible wavelength range as compared to other polymers.-   (2) Reducing the thickness of plastic substrate that is excited by    the laser—The thickness of the material used is limited by the    ability of the structure to withstand the gel filling pressures    which may exceed 350 psig. The biochips used in Examples 5 and 6 are    fabricated by embossing channel features into a 188 micron thick    thermoplastic sheet and then bonding another 188 micron thick    thermoplastic sheet to cover the channels. An experiment to measure    the background fluorescence on glass and plastic substrates was    conducted by placing several substrates (plastic 188 nm, plastic 376    nm, borosilicate glass 0.7 nm, and borosilicate glass 1.4 nm) onto    the separation and detection window of the Genebench optical    detection instrument. (See generally, application Ser. No.    12/080,745, published as 2009/0020427 entitled “Plastic Microfluidic    Separation and Detection Platforms,”) 200 mW of laser excitation was    applied to the substrate and signal was collected. FIG. 79 shows the    background fluorescence of low fluorescence plastic (188 microns and    376 microns thick) and borosilicate glass (0.7 mm and 1.4 mm thick).    The data shows that the background fluorescence for thin films of    plastic is 4 to 7 times lower than that of 0.7 mm and 1.4 mm thick    borosilicate glass.-   (3) Incorporating a filter to reduce background emission—Although    the background fluorescence is low in general, plastic does exhibit    a high autofluorescence at approximately 569 nm. This emission peak    is a result of Raman fluorescence that is generated when plastic is    excited by a 488 nm laser. The spectra of the peak shows that it is    centered about 569 nm and has a full width of 5 nm. The Raman    fluorescence can be eliminated by using a notch filter with a center    wavelength of approximately 570 nm and a rejection band between    approximately 5 to 10 nm. An experiment to assess the improvement in    signal to noise by using a notch filter to eliminate the Raman    plastic fluorescence was conducted. A set of 3 separation and    detection runs was performed without a notch filter, and another set    was performed with a notch filter installed at the input to the PMT    box. FIG. 80 summarizes the results of the data. The data shows that    the filter reduces the peak-to-peak noise for the yellow channel    from 67 to 39 relative fluorescence units (rfu). The filter also    reduces the absolute signal strengths from 1520 to 1236 rfu, with an    overall improvement in the signal to noise ratio from 23 to 32. A    notch filter is useful in the optical systems of both integrated and    unintegrated instruments in settings where the limit of detection is    particularly important (e.g. the analysis of forensic touch samples    and the diagnosis of infectious agents early in the course of a    given disease when pathogen numbers are low).

Example 9 Design of a Fully Integrated Biochip that Purifies NucleicAcids from Clinical Whole Blood Samples, Amplifies the Purified DNA,Sanger Sequences the Amplified DNA, Ultrafilters the Sequenced DNA, andElectrophoretically Separates the Ultrafiltered DNA and GeneratesMultiplexed DNA Sequence Using an Automated Script

Pathogens such as staphylococci, streptococci, and Yersiniaenterocolitica may be present in the extracellular components of theblood. A stationary, unitary plastic biochip that accepts 2 bloodsamples and generates DNA sequence from 8 loci of a given pathogenconsists of a pneumatic-valve-fluidic stack, macrofluidic processingsubassembly, and separation and detection subassembly. The biochipresembles the injection molded biochip of Example 6 with modificationsas described below, and, based on an automated script, the process isconducted as follows:

The macrofluidic subassembly is composed of 12 chambers that holdpreloaded reagents or serve as holding/reaction chambers during the DNApurification process. One chamber is used to accept the blood collectiontube; six chambers are pre-filled with 3 mL of wash solution, 100microliters of cell resuspension solution, 450 microliters of lysissolution, 550 microliters of absolute ethanol, 2 microliters of washbuffer, 2000 microliters of deionized water and 400 microliters ofelution buffer.

The biochipset accepts a standard 3 cc vacutainer tube (for separationexperiments, blood is collected in tubes containing appropriateanticoagulants). The blood collection tube is inserted into the biochipwith the rubber stoppered end down. The purification process isinitiated when the user presses a start button on the instrument. Withinthe instrument, the blood collection tube is pushed onto two hollow pinslocated at the base of blood collection tube cavity. The hollow pinspierce through the rubber stopper, and the blood collection tube ispressurized pneumatically to 5 psi to drive the blood from the bloodcollection tube through a through a filter with nominal pore size of 8microns (such as Leukosorb B media, Pall Corporation, Port Washington,N.Y.) to remove leukocytes from the blood. The flowthrough is passedthrough a through a single layer of 0.2 micron polycarbonate track-etchmembrane (SPI-Pore™ Track-Etch Membrane, Structure Probe, Inc., WestChester, Pa.) to concentrate the bacteria through capture on themembrane, and this flowthrough is routed to a waste chamber.

Resuspension solution (100 microliters) is applied to the surface of thetrack-etch membrane, resuspending the pathogens retained on the tracketch membrane, and generating a concentrated pathogen suspension (whichmay also include residual leukocytes). This suspension is pneumaticallydriven into the lysis chamber. DNA purification is performed asdescribed as in Examples 5 and 6, with all volumes proportionatelylower. Chaotropic lysis reagent is driven into the lysis chamber, andair is pneumatically driven into the lysis/waste chamber to effectchaotic bubbling of the lysate. Ethanol from the ethanol reservoir isdriven into the lysis/waste and mixed by chaotic bubbling. Thelysate/ethanol mixture is pneumatically driven into the holding chamberand through the purification membrane and into the lysis/waste chamber.The membrane is subjected to a series of three washes, removing unboundmaterial and residual lysis solution. The filter is then air-dried. 20.5microliters of elution solution is pneumatically driven from the elutionsolution reservoir through the purification membrane to the eluateholding chamber. This smaller volume ensures that a large proportion ofthe isolated nucleic acids are amplified in the subsequent amplificationreaction.

Eluate is pneumatically driven from the eluate holding chamber into theeluate metering chambers, and excess eluate is pneumatically driven backinto the eluate holding chamber. The eluate is pneumatically driven fromthe eluate metering chamber into the PCR cake chamber. 20.5 microlitersof metered eluate is transferred to reconstitute the cake containing thelyophilized PCR reaction mix to generate the PCR mix for amplification.The cake contains the same components as those of FIGS. 5 and 6, withthe exception that the human STR primer pairs (one of each pair isfluorescently labeled) are replaced with a set of 80 primer pairs(neither member of each pair is labeled). The 80 primer pairs represent8 specific loci for each of 10 pathogens, including staphylococci,streptococci, and Yersinia enterocolitica (much larger sets of loci arepossible if desired). The reconstituted PCR reaction mix ispneumatically driven from the cake chambers into the thermal cyclingchambers and stops at the queuing vent membrane. A thirty-one cycleamplification protocol is applied to cycle the reaction within thethermal cycling chamber. All scripted and process steps are performedessentially as described as in Examples 5 and 6.

PCR product is pneumatically driven from the thermal cycling chambersinto the PCR metering chamber and stops at a vent membrane. Sixmicroliters of the PCR product are mixed with 94 microliters ofdeionized water in a joining chamber. The joining chamber allows themetered PCR and the metered water (which originate from two independentflows) to be combined to form the sample for reconstitution of thelyophilized sequencing cake. The diluted PCR product is driven from thejoining chamber and split into 8 metering chambers, each of which holds11 microliters and are queued by a vent membrane. Each of the eightsamples is driven to a sequencing cake reconstitution chamber. The eightcakes are slightly different versions of the Sanger reaction mix (basedon the use of dye-labeled terminators, such that each extension productbears a single fluorescent label corresponding to the base at thatposition of the sequence), with each cake containing a unique set of tensequencing primers—one for a locus for each of the ten pathogens ofinterest. For a given pathogen, one primer pair for a specific locus isin cake one, a second in cake two, etc.; this placement ensures a singleDNA sequence is generate for a given pathogen from each cake. Thereconstituted sequencing reaction mixes are pneumatically driven fromthe cake chambers into the cycle sequence cycling chambers (located justabove the second thermal cycler) and stop at the queuing vent membrane;each cycling chamber holds 10 microliters. Cycle sequencing is performedas follows: 95° C. for 15 seconds followed by 30 cycles of [95° C. for 5seconds, 50° C. for 10 seconds, and 60° C. for 25 seconds). For each ofeight sequencing reactions (now a total of 16 for two blood samples),the 10 microliter sequencing reaction product is mixed with 100microliters of deionized water in a joining chamber in preparation forultrafiltration.

Electrophoretic separation performance can be greatly improved bypurification of the sequencing product to remove ions necessary forsequencing (and the preceding PCR reaction) that interfere with theseparation. A variety of methods can be employed, includingultrafiltration, in that small ions/primers/unincorporated dye labelsare driven through a filter, leaving the desired product on the filterthat then can be eluted and applied directly to separation anddetection. Ultrafiltration media include polyethersulfone andregenerated cellulose “woven” filters, as well as track-etch membranes,in which pores of highly-uniform size are formed in an extremely thin(1-10 μm) membrane. The latter have the advantage of collecting productof size larger than the pore size on the surface of the filter, ratherthan capturing the product at some depth below the surface.

Accordingly, the diluted sequencing product was driven throughultrafiltration filter. The filter traps Sanger sequencing product butallows ions and the diluted sequencing buffers to pass through. Thematerial on the filter was washed by pneumatically driving 200 μl ofdeionized water through the filter to further remove ions and buffer.Finally the cleaned Sanger sequencing product was eluted from the filterby pneumatically driving 10 μl of deionized water into the filterchamber to resuspend the Sanger sequencing product. The eluate is thenpneumatically driven into the joining chamber. Formamide ispneumatically driven from the formamide reagent chamber into theformamide metering chamber. The formamide flows into the meteringchamber and stops at a vent membrane. Excess formamide is pneumaticallydriven from the formamide chamber into a waste chamber. Ten microlitersof metered ultrafiltered product is pneumatically driven from themetering chamber into the joining chamber. Metered formamide waspneumatically driven from the formamide metering chamber into thejoining chamber. The joining chamber allows the metered ultrafilteredproduct and the metered formamide (which originate from two independentflows) to be combined to form the sample for separation and detection.Electrophoresis is conducted as described in Examples 5 and 6 with theexception that the biochip is heated to 60° C. The injection voltagesand times, and separation voltages and times are identical to that ofExamples 5 and 6. The two blood samples are processed such that theygenerate 16 channels of product for separation and detection. Ingeneral, the number of separation channels per sample is equivalent tothe largest number of loci and primer pairs being interrogated for oneof the pathogens under evaluation.

The optical system is activated to effect laser induced fluorescenceexcitation and detection. The laser is set to 200 mW and the datacollection rate of 5 Hz is implemented. Following color correction, anautomated basecaller generates sequence from lanes that contain one ofthe ten pathogens for which amplification and sequencing primers wereincorporated in the amplification and sequencing cakes, respectively.Approximately 500 bp of sequenced are generated per lane.

The biochip can be modified in many ways, based on the clinical sampletype and the pathogens to be identified. For example, certain bacteriasuch as Francisella tularenis and Chlamydia trachomastis spend asignificant portion of their life cycles within mammalian cells. Someare obligate intracellular organisms and others are optionallyintracellular. The DNA purification process for such intracellularbacteria in blood is similar to that described above with a majorexception. Following application of whole blood on and through the cellseparation filter, the leukocytes trapped by the filter contain the DNAof interest. The filter is washed, resuspended in 100 microliters, andsubjected to guanidinium-based purification as described in Example Iwith corresponding reduction is reagent volume.

If desired, the apparatus can be design to initially lyse the leukocytes(osmotically, for example), taking advantage of the relative ease oflysis of mammalian cells as compared to bacteria. In this setting, theintact intracellular bacteria are released, and the cell extract isbased through a bacterial capture filter and washed. Bacterial DNA isthen purified as described in above. Similarly, whole blood can be lysedin the absence of cell separation, allowing extracellular orintracellular bacterial or viral DNA to be purified.

Example 10 Fully Integrated CNC-Machined and Injection Molded Biochipsthat Store and Release Reagents, Purify Nucleic Acids, Amplify thePurified DNA, Electrophoretically Separate the Amplified DNA andGenerate an STR Profile Using an Automated Script

Biochips of the invention can be designed to incorporate a variety offeatures based on the specific application requirements. For example,another biochip can be constructed by incorporating the single tubeReagent Storage and Release structures of Example 1 into the biochipdesign of Example 5.

A biochip with these features was fabricated by CNC-machiningessentially as described in Example 5. The macrofluidic processingsubassembly is based on same design as that of Example 5. FIG. 47 showsthe macrofluidic block 18 with swab 19, lysis solution 20, ethanol 21,wash solution 22, and elution solution 23 reagent chambers, and fluidicprocessing chambers for lysate holding 24, eluate homogenization 25, air58, TTE 59, and formamide 60 chambers. The lysis solution 20, ethanol21, wash solution 22, and elution solution 23 reagent chambers have theRSR structures of FIG. 1: foil seals (101) were thermally bonded to thetop and bottom of each reagent storage chamber (102). The bottom foilwas bonded first, liquid reagents were filled (indicated by shading of102), and the top foil was then bonded, sealing the reagent storagechamber. The top cover (103) contained pneumatic drive lines thatprovide pressure required to burst the foils.

A second macrofluidic processing assembly (811) (termed the “Matrixloading assembly”; FIG. 81) is included in the biochip. This subassemblycontains sieving matrix (812) and electrophoresis buffer (813), bothwith the RSR structures of FIG. 1. During the initial stages of sampleprocessing, electrophoresis buffer was loaded from the Matrix loadingassembly via the anode of the Separation and Detection subassembly (FIG.55). The foil seals were burst and reagents were released by applyingpneumatic drive pressure of 45 psig to the chambers. The electrophoresisbuffer reagent was then pneumatically driven into the anode chamber at 1psig. Next, sieving matrix was loaded into the electrophoresis channelsvia a hole in the Separation and Detection subassembly (FIG. 55) byapplying a pneumatic drive pressure of 300 psig. Performing sievingmatrix loading in parallel with sample processing (e.g. the nucleic acidpurification) reduces the total time required from sample introductionto STR profile generation. Following matrix loading, pre-electrophoresisis optionally performed.

A photograph of this biochipset, fabricated by CNC-machining (821), isshown in FIG. 82. Lysis solution, ethanol, wash solution, elutionsolution, TTE buffer, and formamide were pre-loaded in the macrofluidicsubassembly (822) and sieving matrix and TTE buffer were pre-loaded inthe matrix loading assembly (811)—all liquid reagents were in chamberswith RSR structures for storage and automated release. Lyophilizedreagents located within the fluidic subassembly included the PCRreaction cake and ILS cake of Example 5 and an allelic ladder cake. Thesample-in to results out process was performed as follows. Five swabswere collected from donors. The swabs were collected by pressing thehead of a Bode SecurSwab swab to the inside of the cheek as instructedby the manufacturer. Each of the 5 swabs was inserted into one of theswab chambers of the biochip. The biochip was inserted to a single,fully integrated instrument as described in Example 5, and the automatedprocess script was initiated to carry out the swab-in to results-outanalysis. The automated script was performed as described in Example 5,with the addition of a step to burst the top and bottom foils of eachliquid reagent chamber and the parallel filling of sieving matrix in thechannels of the Separation and Detection subassembly.

An electropherogram (FIG. 83) was generated for the allelic laddersample (fragments fluorescently labeled with fluorescein, JOE, TAMRA,and ROX). Resolution is single base pair (FIG. 84) as indicated by theclear separation of the Th01 9.3 and 10 alleles shown within a portionof the allelic ladder. The electrophoretic data from one of the buccalswab samples is shown in FIG. 85. Each of fluorescent dyes used to labelthe STR primers (fluorescein, JOE, and TAMRA) in the PCR reaction mixand the ILS (ROX) are shown in individual panels. Fluorescein-labeledSTR fragments are shown in the top panel, JOE-labeled STR fragments areshown in the second panel from the top, TAMRA-labeled STR fragments areshown in the third panel from the top, and ROX-labeled ILS fragments areshown in the bottom panel. The x-axis shows fragment size in base pairsand the Y-axis shows relative fluorescence units. Beneath each fragment,the allele calls are presented in boxes (two calls for heterozygousalleles and one for homozygous alleles). The electrophoretic raw datawas first subjected to color baseline subtraction and smoothing followedby color correction to separate the signals from each of the fluorescentdyes. Following this data processing, the software then performed allelecalling based on using the known fragment sizes of the ILS to assignsizes to the STR peaks in the electropherogram and to assign an allelecall to each sized peak. The results is a full profile, with calledalleles generated from all 16 loci in the multiplexed PCR reaction ofthe fully integrated system. The entire process from insertion ofsamples to generation of the electropherogram required approximately 94minutes, and data processing and allele calling required less than twominutes.

Liquid traps were inserted within the biochipset to prevent the backflow of reagents from the reservoirs into the pneumatic drive lines. Twoforms of liquid traps were used within the biochipset: Dessicat(Molecular sieves) inserted within the macrofluidic processing assemblyare used to absorb liquid that may be present within the pneumatic drivelines while allowing relatively unrestricted air flow. Filter membranes(synthetic fels) were inserted within the SC cover and are used toabsorb liquid from the swab chambers during chaotic bubbling steps whileallowing relatively unrestricted air flow. Vent membranes were also usedto allow air flow through the membranes but halt liquids. Within theinstrument drive lines that can be exposed to liquids (e.g. from anunintentional operator spill of a liquid onto the biochip or instrument)can be protected with a liquid trap between the biochipset and thepneumatic subsystem.

Using the same design as the CNC-machined biochip (including RSRfeatures), an injection molded biochip was fabricated (FIGS. 86-88).FIG. 86 is a photograph of the injection molded biochip, FIG. 87 showsthe top view of the injection molded biochip, and FIG. 88 shows thebottom view of the injection molded biochip. Parts fabricated from COPby injection molding include the pneumatic plate (871), fluidic plate(872), cover to the macrofluidic processing assembly (873), block of themacrofluidic processing assembly (874), spacer plate for themacrofluidic processing assembly (875), cover to the matrix loadingassembly (876), block of the matrix loading assembly (877), and spacerplate for the matrix loading assembly (878). These injection moldedparts were assembled (including thin films, filters, membranes, and RSRcomponents) to form the biochip as described in Examples 1 and 5.

Example 11 A Fully Integrated Biochip that Purifies Bacterial NucleicAcids, Performs Multiplex Amplification on the Purified DNA, andElectrophoretically Separates the Resulting Amplicons to GenerateFragment Sizes and Colors Diagnostic of the Pathogen Using an AutomatedScript

The fully integrated biochips of the invention are capable of analyzinga wide variety of sample types and performing a wide variety of assays,based on the specific design features of the biochip and the compositionof the reagents utilized. Depending on the sample type, features andprocess steps including sample concentration and sample separation intomultiple components (as described in Example 9) may be implemented.

The fully-integrated biochip of Example 5 can accept a variety of sampletypes in addition to buccal swabs. Forensic swabs from crime scenesamples containing blood or epithelial cells (e.g. fingerprints,palmprints, or touch samples in general), clinical swabs (e.g. vaginal,cervical, wound), veterinary swabs, food safety swabs, and environmentalswabs, among others, can be utilized. Tissue samples and liquid samples(including blood, exudate, transudate, urine and other clinical fluids)can be placed in the swab chamber (FIG. 39, 19) for subsequentprocessing.

A fresh overnight culture of B. cereus (ATCC 4342) in 8 ml tryptic soybroth (TSB) media was prepared from a single colony on TSB-agar plate. A1000-fold dilution of the stock homogenized bacteria mixture was used toobtain cell counts under a light microscope using a Petroff-Hausser cellcounter. 100 μl solution containing 10⁵ B. cereus cells was transferredonto a dried human buccal swab. The presence of human cells and DNA onthe swabs models a clinical swab sample in which the vast majority ofDNA is derived from the human patient whereas only a small fraction oftotal nucleic acid is derived from the pathogen or pathogens.

The spiked swab was then placed into the in the swab chamber (FIG. 39,19) of the biochipset of Example 5. Nucleic acid purification,amplification, and separation and detection were performed essentiallyas described in Example 5. The major difference is that the lyophilizedPCR cake located within the PCR cake chamber (FIG. 40, 10) containingprimer pairs that target the 7 house-keeping genes used in B. cereusMLST (see Read, T., et al. (2010). “Rapid multi-locus sequence typingusing microfluidic biochips.” PLoS One 5(5): e10595). The primer pairsused for PCR were designed to amplify fragments of seven B. cereus sensulatu housekeeping genes with sizes ranging from 348-504 base pairs. FIG.89 is the raw data plot of signal strength (RFU) versus S&D run time.Six of the expected loci are present, diagnostic of the presence of B.cereus in the sample. Peaks 1, 2, 3, 4, 5, and 6 are the pur (532 bp),pycA (547 bp), glpF (550 bp), tpi (551 bp), pta (569 bp), and gmk (598bp) amplicons; respectively. The ilvD amplicon is frequently missing inB. cereus strains due to point mutations at a primer site, and itsabsence in the B. cereus strain present in the sample was confirmed byconventional amplification.

The successful identification of a bacterial pathogen in a complexsample can be extended in a variety of ways. In particular, the lysateand eluate volumes can be reduced to generate a more concentrated eluateof purified nucleic acids. These and many related changes (e.g. overallvolume and chamber size reductions, optimization of the thermal cyclingscript) can be utilized to generate single copy level of detection.

Example 12 Fully Integrated Biochips that Store and Release Reagents,Lyse Cells, Directly Amplify DNA Present in the Lysate,Electrophoretically Separate the Amplified DNA and Generate an STRProfile Using an Automated Script

Certain process steps and analogous features can be combined oreliminated to speed sample processing, reduce the size of the biochip,or both. For example, forensic laboratories processing buccal and bloodsamples can eliminate the DNA purification step by lysing cells anddirectly amplifying the lysate. Termed “direct PCR,” this technique issometimes based on the lysis of cells on FTA paper and the inclusion ofadditives in the PCR reaction to minimize inhibition. The AmpFlSTR®Identifiler® Direct PCR Amplification Kit (Applied Biosystems, FosterCity, Calif.), Ampdirect Plus (Whatman, Florham Park, N.J.), and thePowerPlex® 18D System (Promega Corporation, Madison, Wis.) are three ofseveral such products.

The biochipsets of Examples 5, 6, and 10 are modified such thatmacrofluidic processing subassembly eliminates the purification reagentchambers but maintains the lysis solution chamber. Lysis solution isplaced in a dedicated chamber for each sample or is present as a singlechamber and distributed to the five samples. Alternatively, an FTA papersample can be resuspended and placed into the sample chamber,eliminating the need for a lysis reagent. The lysed sample is thendriven to reconstitute the lyophilized PCR cake for direct amplificationfollowed by separation and detection as described in Examples 5, 6, and10. The biochip is fabricated based on the teachings of the presentinvention, and the volume of the macrofluidic processing block isapproximately 80% smaller than those of Examples 5, 6, and 10.

Sample manipulations that can be performed in the biochips of Examples5, 6, and 10 include combinations of nucleic acid extraction; celllysis; cell separation; differential cell lysis; differentialfiltration; total nucleic acid purification; DNA purification; RNApurification; mRNA purification; protein purification; pre-nucleic acidamplification cleanup; nucleic acid amplification (e.g. both singleplexand multiplex end-point PCR, Real-time PCR, reverse transcription PCR,asymmetric PCR, nested PCR, LATE PCR, touchdown PCR, digital PCR,rolling circle amplification, strand displacement amplification, andmultiple displacement amplification); Y-STR amplification; mini-STRamplification; single nucleotide polymorphism analysis; VNTR analysis;RFLP analysis; post-nucleic acid amplification cleanup; pre-nucleic acidsequencing cleanup; nucleic acid sequencing (e.g. Sanger sequencing,pyrosequencing, and single molecule sequencing); post-nucleic acidsequencing cleanup; reverse transcription; pre-reverse transcriptioncleanup; post-reverse transcription cleanup; nucleic acid ligation; SNPanalysis; nucleic acid hybridization; electrophoretic separation anddetection; immunoassays; binding assays; protein assays; enzymaticassays; mass spectroscopy; and nucleic acid and protein quantification.The biochips may combine 2 or more of these sample manipulations, 3 ormore, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 10 or more,12 or more, 15 or more, 20 or more, or 25 or more. The sample splittingdescribed in Example 7 can also be utilized to split a sample into 2 ormore aliquots, with each aliquot being subjected to a unique set ofsample manipulations. The biochips of the invention allow a substantialamount of information to be gained from the interrogation of a givensample.

Although several embodiments of the invention have been described, itwill be apparent to a person skilled in the art that variousmodifications to the details thereof shown and described may be madewithout departing from the scope of the invention.

What is claimed is:
 1. A biochip, which upon insertion into anelectrophoresis instrument having pneumatic, thermal, high voltage, andoptical subsystems, and a process controller, generates a nucleic acidsequencing or sizing profile from at least one sample, said biochipcomprising: a macrofluidic processing subassembly in connection with afluidic subassembly and a pneumatic subassembly, comprising at least onechamber adapted to receive a sample; the fluidic subassembly, comprisinga fluidic plate, and at least one fluid transport channel and anamplification chamber adapted to connecting to the thermal subsystem;the pneumatic subassembly which is adapted to connecting to thepneumatic subsystem of said instrument, and to the subassemblies of saidbiochip, comprising, a pneumatic plate and one or a plurality of drivelines to pneumatically drive fluids on instructions from said processcontroller; a separation and detection subassembly adapted forconnecting to the high voltage and optical subsystems and processcontroller on said instrument, said separation and detection subassemblycomprising a separation channel, and further comprising a detectionregion positioned to send signals from each of said channels to saidoptical subsystem on said instrument; wherein the biochip is plastic,stationary, and unitary, and a footprint of the fluidic and/or pneumaticplates is about 86 mm by 128 mm or greater.
 2. The biochip of claim 1,wherein the footprint of the fluidic and/or pneumatic plates is about100×150 mm or greater.
 3. The biochip of claim 2, wherein the footprintof the fluidic and/or pneumatic plates is about 115×275 mm or greater.4. The biochip of claim 3, wherein the footprint of the fluidic and/orpneumatic plates is about 140×275 or greater.
 5. The biochip of claim 4,wherein the footprint of the fluidic and/or pneumatic plates is 165×295or greater.
 6. A biochip, which upon insertion into an electrophoresisinstrument having pneumatic, thermal, high voltage, and opticalsubsystems, and a process controller, generates a nucleic acidsequencing or sizing profile from at least one sample, said biochipcomprising: a macrofluidic processing subassembly in connection with afluidic subassembly and a pneumatic subassembly, comprising at least onemacrofluidic feature located therein or thereon, further comprising achamber capable of receiving a sample; the fluidic subassembly,comprising a fluidic plate, and at least one feature located therein orthereon, the fluidic subassembly further comprising at least one fluidtransport channel and an amplification chamber adapted to connecting tothe thermal subsystem; the pneumatic subassembly which is adapted toconnecting to the pneumatic subsystem of said instrument, and to thesubassemblies of said biochip, comprising, a pneumatic plate and atleast one feature located therein or thereon, the pneumatic subassemblyfurther comprising one or a plurality of drive lines to pneumaticallydrive fluids on instructions from said process controller; a separationand detection subassembly adapted for connecting to the high voltage andoptical subsystems and process controller on said instrument, saidseparation and detection subassembly comprising at least one featurelocated therein or thereon, the separation and detection subassemblyfurther comprising a separation channel and a detection regionpositioned to send signals from each of said channels to said opticalsubsystem on said instrument; wherein the biochip is plastic,stationary, and unitary, and a physical state of said one or morefeatures of the fluidic, pneumatic, and/or separation and detectionsubassemblies are adapted to change in scripted processes of 25 or moresteps.
 7. Biochip of claim 6, wherein the number of steps is 50 or more.8. Biochip of claim 7, wherein the number of steps is 100 or more. 9.Biochip of claim 8, wherein the number of steps is 200 or more. 10.Biochip of claim 6, wherein the 25 or more scripted process stepsresults in two or more resultant processing steps.
 11. A unitary,stationary biochip, which upon insertion into an electrophoresisinstrument having pneumatic, thermal, high voltage and opticalsubsystems, and a process controller, generates a nucleic acidsequencing or sizing profile from at least one sample, said biochipcomprising: a macrofluidic processing subassembly in connection with afluidic subassembly and a pneumatic subassembly, comprising at least onechamber adapted to receive a sample; the fluidic subassembly, comprisinga fluidic plate having a top and bottom patterned thermoplastic sheetbonded thereon, to form at least one fluid transport channel on eachsides of the plate and an amplification chamber adapted to connecting tothe thermal subsystem; the pneumatic subassembly which is adapted toconnecting to the pneumatic subsystem of said instrument, and to thesubassemblies of said biochip, comprising a pneumatic plate having a toppatterned thermoplastic sheet bonded thereon, and one or a plurality ofdrive lines to pneumatically drive fluids on instructions from saidprocess controller; a valve subassembly, positioned between andconnected to said fluidic and pneumatic subassemblies; and a separationand detection subassembly adapted for connecting to the high voltage andoptical subsystems and process controller on said instrument, saidseparation and detection subassembly comprising at least one separationchannel, and further comprising a detection region positioned to sendsignals from each of said at least one separation channels to saidoptical subsystem on said instrument.
 12. The biochip of claim 11,wherein the separation and detection subassembly is plastic.
 13. Thebiochip of claim 11, in which the macrofluidic, pneumatic, fluidic, andseparation and detection subsystems are plastic.
 14. The biochip ofclaim 11, wherein the valve subassembly comprises at least oneelastomeric valve.
 15. The biochip of claim 11, wherein the valvesubassembly comprises at least one non-elastomeric valve.
 16. Thebiochip of claim 11, wherein the separation and detection subassembly isoriented such that electrophoresis of the sample within the separationand detection subassembly is conducted in the opposite direction from ageneral flow of sample through the fluidic plate.
 17. The biochip ofclaim 11, wherein all reagents needed to process the at least one sampleare contained within the biochip.
 18. The biochip of claim 11, wherein afootprint of the fluidic plate and/or the pneumatic plate is about 86mm×128 mm or greater.
 19. The biochip of claim 11 wherein a footprint ofthe fluidic plate and/or the pneumatic plate is about 115 mm×275 mm orgreater.
 20. The biochip of claim 11 wherein a footprint of the fluidicplate and/or the pneumatic plate is about 165 mm×295 mm or greater.